Composition of fermented product and nasturtium root extract and application thereof in preparing skin care products

Abstract

The present invention relates to the technical field of plant extracts, and in particular to a composition of a fermented product and a Herba Lycopodii Extract and an application thereof in the preparation of skin care products. The present invention provides a composition of a fermentation filtrate of Bifidobacterium longum subsp. Infantis and a Herba Lycopodii Extract; experiments show that the composition formed by the two can effectively remove DPPH free radicals, resist cell damage caused by UVA, downregulate the expression of aging-related genes and proteins, upregulate the expression of anti-aging genes, and inhibit the activity of tyrosinase, and has good whitening and anti-aging effects. After the two are compounded in a specific ratio, obvious synergistic whitening and anti-aging effects can be produced, and the composition has a good application prospect in skin care products and health care products.

Description

Technical Field

[0001] The invention relates to the technical field of plant extracts, and in particular to a composition of a fermentation product and a nephrite extract and application thereof in preparing skin care products. Background Art

[0002] Anti-aging and whitening have always been the two key goals that consumers are constantly pursuing, and they are the main line of skin care demands for people of all ages and skin types. Skin aging is not only manifested in changes in appearance such as wrinkles, sagging, and sagging, but is also related to slowed skin metabolism, water loss, and weakened barrier function; while skin pigmentation greatly affects the uniformity of facial color and overall appearance.

[0003] At present, there are many kinds of products with whitening effect on the market, but some of them cannot have both whitening and anti-aging effects, and the effect is single, or the whitening and anti-aging effects are not ideal. Therefore, it is of great value and significance to develop a product with significant whitening and anti-aging effects and few side effects.

[0004] The ground nasturtium is a large perennial clumping herb of the Musaceae family. It has a long history of being used as a herbal medicine. Its pharmacological effects include hemostasis, anti-inflammatory, antibacterial, and anti-tumor.

[0005] At present, there is no research or report on using the combination of the Herba Lysimachiae extract and probiotics for whitening and anti-aging. Summary of the invention

[0006] In view of this, the present invention provides a Herba Lysimachiae extract and application thereof in preparing antioxidant, anti-aging and whitening products.

[0007] The composition provided by the invention comprises a nephrolepis radix extract and a fermentation filtrate of Bifidobacterium longum.

[0008] The present invention compounded the extract of Herba Lycopodii and the fermentation filtrate of Bifidobacterium longum infantis subsp. Studies have shown that the whitening and anti-aging effects of the composite obtained by compounding the two are significant. Under a specific ratio, the two components have an obvious synergistic effect. In addition, the present invention also attempts to compound other strains (such as bifid yeast) with the Herba Lycopodii extract, and it is found that the effect is obviously not as good as the composition of the present invention.

[0009] In the composition of the present invention, the mass ratio of the Bifidobacterium longum fermentation broth and the Herba Lycopodii extract is (1~100):(100:1), specifically 100:1, 10:1, 1:1, 1:10 or 1:100, and also includes any other mass ratio and mass ratio range within the above range.

[0010] In the composition of the present invention, the Bifidobacterium longum includes Bifidobacterium longum subsp. infantis. Preferably, the Bifidobacterium longum subsp. infantis; in some specific embodiments, the deposit number of the Bifidobacterium longum subsp. infantis is CGMCC NO.27851.

[0011] In a specific embodiment of the present invention, the Bifidobacterium longum fermentation broth is Bifidobacterium longum subspecies infantis fermentation broth, and the preparation method refers to Chinese patent application number 202410036419.6, comprising the following steps:

[0012] (1) Preparation of glycerol tube strains

[0013] 1) Activation of the original lyophilized powder: transfer the lyophilized powder to 100 mL of Huankai MRS triangular flask culture medium, and culture at 37℃ for 20-24 hours to prepare bacterial solution for use;

[0014] 2) Isolate single colonies: Streak the bacterial liquid on MRS solid culture medium plates, culture at 37℃ for 48 hours, and keep them for later use after they grow up;

[0015] 3) Activation of single colony: transfer single colony to 100 mL liquid Erlenmeyer flask culture medium, and activate the bacterium-making liquid by anaerobic static culture at 37℃ for 20 hours. Check under a microscope to see if OD*10>0.260 and pH<4.50 meet the standards;

[0016] 4) Preservation of original glycerol tube strains: 5% inoculation of the above bacteria (OD*10>0.260 after 20-24 hours of culture can be considered qualified) is transferred to the culture medium in the triangular flask of formula card 1, and OD*10>0.260 and pH<4.50 are detected after 20 hours of culture. After the microscopic examination meets the standards, the strains are preserved in glycerol tubes (0.70 mL of preservative solution and 0.70 mL of bacterial solution).

[0017] 5) Establishment of seed batches: The seed batch system for production strains should be established in accordance with the relevant provisions of "Management and Quality Control of Strains for Production and Inspection of Biological Products".

[0018] (2) The strain Bifidobacterium longum subsp. infantis obtained in step (1) was inoculated into the culture medium with an inoculum amount of 5% (the culture was qualified if OD*10>0.260 was detected after 20-24 hours of culture), and cultured at 37°C for 20-24 hours. The supernatant was obtained by centrifugation and freeze-dried to obtain a concentrated fermentation liquid;

[0019] The culture medium involved in the previous preparation method includes: 12 g / L lactose, 10 g / L wheat peptone, 10 g / L yeast powder, 2 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 2 g / L triammonium citrate, 0.58 g / L magnesium sulfate 7hydrate, 0.05 g / L manganese sulfate and 2 g / L Tween 80.

[0020] In the composition of the present invention, the extract of the ground nasturtium is the extract of the ground nasturtium and its flower buds.

[0021] In the composition of the present invention, the preparation method of the extract of the nephrolepis terrestris comprises: taking the flowers and buds of the nephrolepis terrestris, cold-immersing and extracting them with 75% ethanol, and concentrating them to dryness under reduced pressure to obtain a crude extract of the nephrolepis terrestris.

[0022] The present invention also provides the use of the composition in preparing products with whitening and anti-aging effects.

[0023] In the present invention, free radical oxidation and UVA damage are both important causes of aging. In the present invention, the composition achieves anti-aging effects by resisting oxidation, UVA damage and regulating the levels of genes and proteins related to anti-aging.

[0024] In some embodiments, the present invention has been found through research that the composition of the present invention can effectively scavenge DPPH free radicals and exert anti-aging effects through antioxidant effects.

[0025] In some embodiments, the present invention has found through research that the composition of the present invention can effectively improve the cell viability after UVA stimulation and has a significant resistance to UVA damage. Specifically, the cells are fibroblasts.

[0026] MMP1 is a matrix metalloproteinase, and its main function is to decompose interstitial collagen, including types I, II and III. Collagen is the main structural protein in the dermis of the skin. When collagen is degraded, the mechanical support of the skin is lost, which leads to the appearance of wrinkles. MMP3 is a matrix metalloproteinase III, which can degrade some protein components in the extracellular matrix, such as collagen. In the skin, the degradation of collagen is related to skin aging. Some studies have shown that MMP1 and MMP3 help improve the elasticity and firmness of the skin. Therefore, inhibiting MMP1 and MMP3 can achieve a certain effect in resisting the formation of wrinkles and exert anti-aging effects.

[0027] COL1 is type I collagen, which accounts for about 80% of the dermis. Type I collagen helps the dermis (middle layer of the skin) form fibroblasts, helps new cells grow, provides structure, strength and support for the skin, and makes the skin plump and full. COL1 gene The expression of can resist the formation of wrinkles to a certain extent, thus achieving the effect of anti-aging.

[0028] In some embodiments, the present invention has been found through research that the composition of the present invention can inhibit MMP1, MMP3 Gene transcription, inhibiting the expression of MMP1 protein and promoting COL1 , COL3 Transcription of genes.

[0029] The present invention also provides a product with whitening and anti-aging effects, comprising the above-mentioned composition.

[0030] In the product of the present invention, the concentration of the fermentation filtrate of Bifidobacterium longum is 0.002mg / ml~10mg / ml, preferably 0.002mg / ml~0.02mg / ml, 0.002mg / ml~0.1mg / ml, 0.002mg / ml~0.2mg / ml, 0.02mg / ml~0.1mg / ml, 0.02mg / ml~0.2mg / ml, 0.1mg / ml~1mg / ml, 0.1mg / ml~5mg / ml, 0.1mg / ml~10mg / ml, 1mg / ml~5mg / ml, 1mg / ml~10mg / ml or 5mg / ml~10mg / ml; specifically 0.002mg / ml, 0.02mg / ml, 0.1mg / ml, 0.2mg / ml, 1mg / ml, 5mg / ml or 10mg / ml.

[0031] In the product of the present invention, the concentration of the Herba Lycoris Radiatae Extract is 0.002mg / ml~10mg / ml, preferably 0.002mg / ml~0.02mg / ml, 0.002mg / ml~0.1mg / ml, 0.002mg / ml~0.2mg / ml, 0.02mg / ml~0.1mg / ml, 0.02mg / ml~0.2mg / ml, 0.1mg / ml~1mg / ml, 0.1mg / ml~5mg / ml, 0.1mg / ml~10mg / ml, 1mg / ml~5mg / ml, 1mg / ml~10mg / ml or 5mg / ml~10mg / ml; specifically 0.002mg / ml, 0.02mg / ml, 0.1mg / ml, 0.2mg / ml, 1mg / ml, 5mg / ml or 10mg / ml.

[0032] In the present invention, the dosage form of the product includes skin care products or medicines; the dosage form of the skin care products includes but is not limited to creams, lotions, solutions, sprays, gels or powders; the dosage form of the medicines includes tablets, emulsions, granules, pills, liquid preparations, ointments or patches.

[0033] The present invention provides Bifidobacterium longum subsp. infantis (Bifidobacterium longum subsp. Infantis) and a composition of a phytohormone extract; experiments show that the composition formed by the two can effectively remove DPPH free radicals, resist cell damage caused by UVA, downregulate the expression of aging-related genes and proteins, upregulate the expression of anti-aging genes, and inhibit the activity of tyrosinase, and has good whitening and anti-aging effects. After the two are compounded in a specific ratio, they can produce obvious synergistic effects, and have a good application prospect in skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The DPPH free radical scavenging rate of filtrates with different concentrations is shown;

[0035] Figure 2 The DPPH free radical scavenging rate of crude extracts of Hedyssa suberecta at different concentrations is shown;

[0036] Figure 3 The DPPH free radical scavenging rate of different ratios of the filtrate and the crude extract of the Herba Lysimachiae was shown;

[0037] Figure 4 The cell viability of filtrates with different concentrations is shown;

[0038] Figure 5 The cell survival rates of crude extracts of Hedyssa subspinipes at different concentrations are shown;

[0039] Figure 6 The cell survival rates of different ratios of the filtrate and the crude extract of Herba Lycoris Radiatae were shown;

[0040] Figure 7 The filtrate of different concentrations MMP1 Relative mRNA levels of genes;

[0041] Figure 8 The filtrate of different concentrations MMP3 Relative mRNA levels of genes;

[0042] Fig. 9 The filtrate of different concentrations COL1 Relative mRNA levels of genes;

[0043] Fig.10 The filtrate of different concentrations COL3 Relative mRNA levels of genes;

[0044] Fig.11 The results show that different concentrations of crude extract of Hedysarum sphaerocephalum MMP1 Relative mRNA levels of genes;

[0045] Fig.12 The results show that different concentrations of crude extract of Hedysarum sphaerocephalum MMP3 Relative mRNA levels of genes;

[0046] Fig.13 The results show that different concentrations of crude extract of Hedysarum sphaerocephalum COL1 Relative mRNA levels of genes;

[0047] Fig.14 The results show that different concentrations of crude extract of Hedysarum sphaerocephalum COL3 Relative mRNA levels of genes;

[0048] Fig.15 The results of different ratios of filtrate and crude extract of Hedysarum sphaerocephalum were shown in Figure 2. MMP1 Relative mRNA levels of genes;

[0049] Fig.16 The results of different ratios of filtrate and crude extract of Hedysarum sphaerocephalum were shown in Figure 2. MMP3 Relative mRNA levels of genes;

[0050] Fig.17 The results of different ratios of filtrate and crude extract of Hedysarum sphaerocephalum were shown in Figure 2. COL1 Relative mRNA levels of genes;

[0051] Fig.18 The results of different ratios of filtrate and crude extract of Hedysarum sphaerocephalum were shown in Figure 2. COL3 Relative mRNA levels of genes;

[0052] Fig.19 The relative expression levels of MMP1 protein in the filtrate, the crude extract of Herba Lycoris Radiatae and different combinations are shown;

[0053] Fig. 20 The tyrosinase inhibition rate of filtrates with different concentrations is shown;

[0054] Fig.21 The tyrosinase inhibition rate of crude extracts of Hedysarum terrestris at different concentrations is shown;

[0055] Fig. 22 The tyrosinase inhibition rates of different ratios of the filtrate and the crude extract of Nymphaeaceae are shown. DETAILED DESCRIPTION

[0056] The present invention provides a composition of a fermented product and a Herba Lycopodii extract 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 is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all 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.

[0057] 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.

[0058] 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.

[0059] “And / or” as used herein includes the meanings of “and,” “or,” and “all or any other combination of elements linked by the term.”

[0060] 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.

[0061] It should be understood that in various embodiments of the present invention, serial numbers do 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.

[0062] 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.

[0063] Some cases are recorded in the embodiments and comparative examples of the present invention. The embodiments show some implementations 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 good effects. In the cases with better effects, the mass ratios of fermentation filtrate and crude extract of Herba Lycoris Radiatae are 1:100, 1:10, 1:1, 10:1, and 100:1. The present invention also attempts to use different components, different ratios, and different strains to carry out anti-aging and whitening experiments, but the effects of these schemes are not as good as the effects under the ratios described in the present invention, and will not be repeated here one by one.

[0064] 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.

[0065] In the embodiments of the present invention, the synergistic effect analysis adopts the Bliss independence model. The Bliss independence model is used to simulate the effects of two or more single drugs in combination, and is a common and reliable model for calculating drug synergy coefficients in the field. For relevant 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".

[0066] The Bliss synergy score was calculated according to the Bliss independence model, with the formula: 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, SBLISS >0 indicates synergistic effect.

[0067] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0068] In the present invention, unless otherwise specified, "filtrate" refers to the fermentation filtrate of Bifidobacterium longum subsp. infantis. "Crude extract of nasturtium troglodytes" refers to the ethanol extract of nasturtium troglodytes and flower buds.

[0069] Bifidobacterium longum subsp. infantis ( Bifidobacterium longum subsp. Infantis )SGBifido001, deposited on July 10, 2023 at the General Microbiology Center of China Culture Collection Administration; address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; deposit number: CGMCCNo.27851. This subspecies has been disclosed in the Chinese patent application number 202410036421.3.

[0070] The preparation of the fermentation filtrate of Bifidobacterium longum subsp. infantis comprises:

[0071] (1) Preparation of glycerol tube strains

[0072] 1) Activation of the original lyophilized powder: transfer the lyophilized powder to 100 mL of Huankai MRS triangular flask culture medium, and culture at 37℃ for 20-24 hours to prepare bacterial solution for use;

[0073] 2) Isolate single colonies: Streak the bacterial liquid on MRS solid culture medium plates, culture at 37℃ for 48 hours, and keep them for later use after they grow up;

[0074] 3) Activation of single colony: transfer single colony to 100 mL liquid Erlenmeyer flask culture medium, and activate the bacterium-making liquid by anaerobic static culture at 37℃ for 20 hours. Check under a microscope to see if OD*10>0.260 and pH<4.50 meet the standards;

[0075] 4) Preservation of original glycerol tube strains: 5% inoculation of the above bacteria (OD*10>0.260 after 20-24 hours of culture can be considered qualified) is transferred to the culture medium in the triangular flask of formula card 1, and OD*10>0.260 and pH<4.50 are detected after 20 hours of culture. After the microscopic examination meets the standards, the strains are preserved in glycerol tubes (0.70 mL of preservative solution and 0.70 mL of bacterial solution).

[0076] 5) Establishment of seed batches: The seed batch system for production strains should be established in accordance with the relevant provisions of "Management and Quality Control of Bacterial and Viral Strains for Production and Inspection of Biological Products".

[0077] (2) The strain Bifidobacterium longum subsp. infantis obtained in step (1) was inoculated into the culture medium with an inoculum amount of 5% (the culture was qualified if OD*10>0.260 was detected after 20-24 hours of culture), and cultured at 37°C for 20-24 hours. The supernatant was obtained by centrifugation and freeze-dried to obtain a concentrated fermentation liquid;

[0078] The culture medium involved in the above preparation method includes: 12 g / L lactose, 10 g / L wheat peptone, 10 g / L yeast powder, 2 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 2 g / L triammonium citrate, 0.58 g / L magnesium sulfate 7hydrate, 0.05 g / L manganese sulfate and 2 g / L Tween 80.

[0079] The present invention will be further described below in conjunction with embodiments: Example 1

[0080] 1. DPPH free radical scavenging rate test to determine the antioxidant effect

[0081] 1.1 Determination of DPPH free radical scavenging rate of samples

[0082] (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:

[0083] (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.

[0084] (3) The positive control substance Trolex was diluted to 0.025 mM with pure water to verify the test system.

[0085] (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).

[0086] (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).

[0087] (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.

[0088] (7) Let the reaction stand at room temperature for 5 minutes and measure OD 517 .

[0089] (8) Calculate the DPPH free radical scavenging rate: scavenging rate (%) = (1- )×100%.

[0090] (9) In the formula, T is the absorbance of the sample well, that is, 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, that is, the absorbance of the DPPH solution when no sample is added; and C0 is the background absorbance of the solvent.

[0091] 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 intra-group comparison before and after the experiment, and one-way analysis of variance was used for comparison of means between groups. P<0.05 indicated that the difference was statistically significant.

[0092] Table 1 DPPH free radical scavenging rate test sample solution table

[0093] The test results are shown in Tables 2~4 and Figures 1 to 3 .

[0094] 1.2 Antioxidant efficacy of the filtrate (DPPH free radical scavenging rate)

[0095] Table 2 DPPH free radical scavenging rate of filtrates at different concentrations

[0096] Summary: The positive standard 0.025mM Trolex showed a good DPPH free radical scavenging rate, indicating that the experimental system was successful. The filtrates of different concentrations showed excellent DPPH free radical scavenging rates, and the 10mg / ml filtrate had a DPPH free radical scavenging rate of 50%, indicating that the filtrate had a strong antioxidant capacity and a strong anti-aging effect.

[0097] 1.3 Antioxidant effect of crude extract of N. terrestris (DPPH free radical scavenging rate)

[0098] Table 3 DPPH free radical scavenging rate of crude extracts of different concentrations of Hedyssa suberecta

[0099]

[0100] Summary: The use of 0.025mM Trolex showed a good DPPH free radical scavenging rate, indicating that the experimental system was successful. Different concentrations of the crude extract of the ground jellyfish showed excellent DPPH free radical scavenging rates. The DPPH free radical scavenging rate of the crude extract of the ground jellyfish at 1mg / ml exceeded 90%, indicating that the crude extract of the ground jellyfish has a strong antioxidant capacity and a strong anti-aging effect.

[0101] 1.4 Antioxidant synergistic effect of different ratios of filtrate + crude extract of Nasturtium sutchuenensis (DPPH free radical scavenging rate)

[0102] Table 4 DPPH free radical scavenging rate of different ratios of filtrate and crude extract of Hedysarum sphaerocephalum

[0103]

[0104] Conclusion: Different ratios of filtrate and crude extract of Hedysarum sutchuenensis showed excellent DPPH free radical scavenging rate. When the ratio of filtrate: crude extract of Hedysarum sutchuenensis was 100:1, the DPPH free radical scavenging rate increased significantly compared with the single filtrate and crude extract of Hedysarum sutchuenensis. BLISS>0, indicating that the filtrate and the crude extract of Nasturtium schrenkiana have strong antioxidant capacity at the combination of 100:1, and the filtrate and the crude extract of Nasturtium schrenkiana have a synergistic anti-aging effect.

[0105] 2. MTT assay to determine cell viability

[0106] 2.1 Determination of cell viability

[0107] (1) HFF fibroblasts were plated into 96-well plates.

[0108] (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.

[0109] (3) Discard the culture medium and add MTT solution (0.5 mg / mL) to each well at 37°C and incubate 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:

[0110] Cell viability (%) =100%×

[0111] The results are shown in Tables 5~7 and Figures 4 to 6 .

[0112] 2.2 Filtrate against UVA damage (MTT)

[0113] Table 5 Numerical table of cell viability of filtrates of different concentrations

[0114]

[0115] 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 filtrate, the detected cell viability increased significantly compared with UVA treatment alone, indicating that the filtrate has the effect of resisting cell damage caused by UVA and has anti-aging effects.

[0116] 2.3 Effect of extract of Herba Lycoris Radiatae against UVA damage (MTT)

[0117] Table 6 Numerical table of cell viability of crude extracts of different concentrations of Hedysarum sphaerocephalum

[0118]

[0119] 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 crude extract of Geum schrenkiana, the detected cell viability increased to a certain extent compared with UVA treatment alone, indicating that the crude extract of Geum schrenkiana has the effect of resisting cell damage caused by UVA and has anti-aging effects.

[0120] 2.4 Synergistic effect of different ratios of filtrate + crude extract of Herba Lycoris Radiatae against UVA damage (MTT)

[0121] Table 7 Cell viability values ​​of different ratios of filtrate and crude extract of Hedysarum sphaerocephalum

[0122]

[0123] Summary: After UVA treatment, the cell viability decreased significantly, indicating that UVA stimulation caused damage to the cell survival rate, and the anti-aging model was successfully established; under UVA stimulation, the filtrate, the crude extract of the scutellaria baicalensis and the combination were used to treat and count the cell survival rate. The results showed that when the ratio of the filtrate: the crude extract of the scutellaria baicalensis was 100:1, the cell survival rate increased significantly compared with the single filtrate and the crude extract of the scutellaria baicalensis.

[0124] 3 Detection and analysis of anti-aging genes (qPCR)

[0125] 3.1 qPCR determination of anti-aging gene expression

[0126] (1) HFF fibroblasts grown in 12-well plates were stimulated with UVA to establish the model.

[0127] (2) After modeling, medication was administered according to the dosing schedule in Table 8, and cells were collected 24 hours later.

[0128] (3) Use an RNA total extraction kit to extract cellular RNA, and use a reverse transcription kit to reverse transcribe and synthesize cDNA.

[0129] (4) Amplify using SYBR Green qPCR Master Mix fluorescence quantitative PCR instrument and check the specificity of primers through melting curve.

[0130] (5) Calculate the target gene (type I matrix metalloproteinase) using the 2^-ΔΔCt method MMP1 , type III matrix metalloproteinase MMP3 , collagen type I COL1 and type III collagen COL3 ) relative expression levels, GAPDH As an internal reference gene ( GAPDHPrimer sequences: F: 5′-ACAACTTTGGTATCGTGGAAGG-3′; R: 5′-GCCATCACGCCACAGTTTC-3′).

[0131] 3.2 Anti-aging effect of filtrate (qPCR)

[0132] Table 8 qPCR experimental design table

[0133]

[0134] The results are shown in Tables 9 to 12 and Figures 7 to 10 .

[0135] (1) MMP1 PCR

[0136] Matrix metallopeptidase 1 (MMP1) Primers:

[0137] F:5'-AAAATTACACGCCAGATTTGCC-3';

[0138] R:5'-GGTGTGACATTACTCCAGAGTTG-3'.

[0139] MMP1 is a matrix metalloproteinase, which mainly decomposes interstitial collagen, including types I, II and III. Collagen is the main structural protein in the dermis of the skin. When collagen is degraded, the mechanical support of the skin is lost, which leads to the appearance of wrinkles. MMP1 Changes in gene expression are used to evaluate the anti-wrinkle efficacy of the test substance.

[0140] Table 9 Filtrates of different concentrations MMP1 Table of relative mRNA levels of genes

[0141]

[0142] Conclusion: After UVA treatment, compared with BC, MMP1 The relative mRNA level of the gene was significantly upregulated, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used. MMP1 The relative mRNA level of the gene decreased significantly, indicating that the positive control of the experiment was effective. Compared with the UVA alone treatment group, after treatment with 0.002mg / ml and 0.1mg / ml filtrate, MMP1 The relative mRNA level of the gene decreased significantly, indicating that the filtrate can significantly inhibit MMP1 The expression of genes has certain anti-aging effects.

[0143] 2) MMP3 PCR

[0144] Matrix metallopeptidase 3 (MMP3) Primers:

[0145] F:5'-CTGGACTCCGACACTCTGGA-3'

[0146] R:5'-CAGGAAAGGTTCTGAAGTGACC-3'

[0147] MMP3 is a matrix metalloproteinase III that can degrade some protein components in the extracellular matrix, such as collagen. In the skin, the degradation of collagen is associated with skin aging. Some studies have shown that MMP3 may play a role in skin tissue remodeling, helping to improve skin elasticity and firmness by promoting the repair and regeneration of the extracellular matrix. In addition, MMP3 may be involved in regulating the proliferation and migration of skin cells. This is essential for the repair and regeneration process of the skin. After the test substance is applied to the cell model, it is tested by MMP3 Changes in gene expression are used to evaluate the anti-wrinkle efficacy of the test substance.

[0148] Table 10 Filtrates of different concentrations MMP3 Table of relative mRNA levels of genes

[0149]

[0150] Conclusion: After UVA treatment, compared with BC, MMP3 The relative mRNA level of the gene was significantly upregulated, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used. MMP3 The relative mRNA level of the gene decreased significantly, indicating that the positive control of the experiment was effective. Compared with the UVA alone treatment group, after 0.02 mg / ml filtrate treatment, MMP3 The relative mRNA level of the gene decreased significantly, indicating that the filtrate concentration of 0.02 mg / ml can significantly inhibit the intracellular MMP3 The expression of genes has certain anti-aging effects.

[0151] (3) COL1 PCR

[0152] Collagen I (COL1) Primers:

[0153] F:5'- GTGCGATGACTGATCTGTGA-3'

[0154] R:5'- CGGTGGTTTCTTGGTCGGT-3'

[0155] COL1 is type I collagen, which accounts for about 80% of the dermis. Type I collagen helps the dermis (middle layer of the skin) form fibroblasts, helps new cells grow, provides structure, strength and support for the skin, makes the skin plump and full, and promotes COL1 The expression of protein can resist the formation of wrinkles to a certain extent, thus achieving the anti-aging effect.

[0156] Table 11 Filtrates of different concentrations COL1 Table of relative mRNA levels of genes

[0157]

[0158] Conclusion: After UVA treatment, compared with BC, COL1 The relative mRNA level of the gene decreased significantly, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used. COL1 The relative mRNA level of the gene increased significantly, indicating that the positive control of the experiment was effective. Compared with the UVA alone treatment group, after treatment with different concentrations of filtrate, COL1 The relative mRNA level of the gene was significantly increased, indicating that the filtrate can significantly increase the intracellular COL1 The expression of genes has certain anti-aging effects.

[0159] (4) COL3 PCR

[0160] Collagen III (COL3) Primers:

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

[0162] R: 5'-ACAGACACATATTTGGCATGGTT-3

[0163] 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.

[0164] Table 12 Filtrates of different concentrations COL3 Table of relative mRNA levels of genes

[0165]

[0166] Conclusion: After UVA treatment, compared with BC, COL3 The relative mRNA level of the gene decreased significantly, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used. COL3 The relative mRNA level of the gene increased significantly, indicating that the positive control of the experiment was effective. Compared with the UVA-only treatment group, after treatment with 0.02 mg / ml and 0.1 mg / ml concentrations of filtrate, COL3 The relative mRNA level of the gene was significantly increased, indicating that the filtrate concentration of 0.02mg / ml and 0.1mg / ml can significantly increase the intracellular COL3 The expression of genes has certain anti-aging effects.

[0167] 3.3 Anti-aging effect of crude extract of Nasturtium schrenkiana (qPCR)

[0168] Table 13 qPCR experimental design table

[0169]

[0170] The test results are shown in Tables 14 to 17 and Figures 11 to 14 .

[0171] (1) MMP1 PCR

[0172] Matrix metallopeptidase 1 (MMP1) Primers:

[0173] F:5'-AAAATTACACGCCAGATTTGCC-3'

[0174] R:5'-GGTGTGACATTACTCCAGAGTTG-3'

[0175] MMP1 is a matrix metalloproteinase, which mainly decomposes interstitial collagen, including types I, II and III. Collagen is the main structural protein in the dermis of the skin. When collagen is degraded, the mechanical support of the skin is lost, which leads to the appearance of wrinkles. MMP1 Changes in gene expression are used to evaluate the anti-wrinkle efficacy of the test substance.

[0176] Table 14 Different concentrations of crude extracts of Hedysarum truncatum MMP1 Table of relative mRNA levels of genes

[0177]

[0178] Conclusion: After UVA treatment, compared with BC, MMP1 The relative mRNA level of the gene was significantly upregulated, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used. MMP1The relative mRNA level of the gene decreased significantly, indicating that the positive control of the experiment was effective. Compared with the UVA treatment group, after treatment with different concentrations of the crude extract of the Herba Lycopodii, MMP1 The relative mRNA level of the gene decreased significantly, indicating that the crude extract of Hedysarum sutchuenensis can significantly inhibit MMP1 The expression of genes has significant anti-aging effects.

[0179] (2) MMP3 PCR

[0180] Matrix metallopeptidase 3 (MMP3) Primers:

[0181] F:5'-CTGGACTCCGACACTCTGGA-3'

[0182] R:5'-CAGGAAAGGTTCTGAAGTGACC-3'

[0183] MMP3 is a matrix metalloproteinase III that can degrade some protein components in the extracellular matrix, such as collagen. In the skin, the degradation of collagen is associated with skin aging. Some studies have shown that MMP3 may play a role in skin tissue remodeling, helping to improve skin elasticity and firmness by promoting the repair and regeneration of the extracellular matrix. In addition, MMP3 may be involved in regulating the proliferation and migration of skin cells. This is essential for the repair and regeneration process of the skin. After the test substance is applied to the cell model, it is tested by MMP3 Changes in gene expression are used to evaluate the anti-wrinkle efficacy of the test substance.

[0184] Table 15 Different concentrations of crude extracts of Hedysarum truncatum MMP3 Table of relative mRNA levels of genes

[0185]

[0186] Conclusion: After UVA treatment, compared with BC, MMP3 The relative mRNA level of the gene was significantly upregulated, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used. MMP3 The relative mRNA level of the gene decreased significantly, indicating that the positive control of the experiment was effective. Compared with the UVA treatment group alone, after treatment with 0.1mg / ml and 0.2mg / ml of the crude extract of the Herba Lysimachiae, MMP3 The relative mRNA level of the gene decreased significantly, indicating that the crude extract of the genus Nymphaeaceae at a concentration of 0.1 mg / ml and 0.2 mg / ml can significantly inhibit the intracellular MMP3 The expression of genes has certain anti-aging effects.

[0187] (3) COL1PCR

[0188] Collagen I (COL1) Primers:

[0189] F:5'- GTGCGATGACTGATCTGTGA-3'

[0190] R:5'- CGGTGGTTTCTTGGTCGGT-3'

[0191] 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.

[0192] Table 16 Different concentrations of crude extracts of Hedysarum truncatum COL1 Table of relative mRNA levels of genes

[0193]

[0194] Conclusion: After UVA treatment, compared with BC, COL1 The relative mRNA level of the gene decreased significantly, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used. COL1 The relative mRNA level of the gene increased significantly, indicating that the positive control of the experiment was effective. Compared with the UVA treatment group, after treatment with different concentrations of the crude extract of the Herba Lycopodii, COL1 The relative mRNA level of the gene was significantly increased, indicating that the crude extract of Hedysarum sibiricum can significantly increase the COL1 The expression of genes has certain anti-aging effects.

[0195] (4) COL3 PCR

[0196] Collagen III (COL3) Primers:

[0197] F: 5'-TTGAAGGAGGATGTTCCCATCT-3'

[0198] R: 5'-ACAGACACATATTTGGCATGGTT-3

[0199] 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.

[0200] Table 17 Different concentrations of crude extracts of Hedysarum truncatum COL3 Table of relative mRNA levels of genes

[0201]

[0202] Conclusion: After UVA treatment, compared with BC, COL3 The relative mRNA level of the gene decreased significantly, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used. COL3 The relative mRNA level of the gene increased significantly, indicating that the positive control of the experiment was effective. Compared with the UVA treatment group, after treatment with 0.02 mg / ml concentration of the crude extract of the Herba Lycoris Radiatae, COL3 The relative mRNA level of the gene was significantly increased, indicating that the crude extract of the ground lotus at a concentration of 0.02 mg / ml can significantly increase the intracellular COL3 The expression of genes has certain anti-aging effects.

[0203] 3.4 Filtrate + Rhizoma Dioscoreae Extract Anti-aging Synergistic Effect (qPCR)

[0204] Table 18 qPCR experimental design table

[0205]

[0206] The test results are shown in Tables 19 to 22 and Figures 15 to 18 。

[0207] (1) MMP1 PCR

[0208] Matrix metallopeptidase 1 (MMP1) Primers:

[0209] F:5'-AAAATTACACGCCAGATTTGCC-3';

[0210] R:5'-GGTGTGACATTACTCCAGAGTTG-3'.

[0211] MMP1 is a matrix metalloproteinase, which mainly decomposes interstitial collagen, including types I, II and III. Collagen is the main structural protein in the dermis of the skin. When collagen is degraded, the mechanical support of the skin is lost, which leads to the appearance of wrinkles. MMP1 Changes in gene expression are used to evaluate the anti-wrinkle efficacy of the test substance.

[0212] Table 19 Combinations of filtrate and crude extract of Hedysarum truncatum in different proportions MMP1 Table of relative mRNA levels of genes

[0213]

[0214] Conclusion: After UVA treatment, compared with BC, MMP1 The relative mRNA level of the gene increased significantly, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used. MMP1 The relative mRNA level of the gene decreased significantly, indicating that the positive control of the experiment was effective. The filtrate and the crude extract of the ground jellyfish were combined at a ratio of 10:1 and 100:1. MMP1 The gene expression levels of S BLISS >0, indicating that the filtrate and the crude extract of Nymphaeaceae have a synergistic anti-aging effect, indicating that the filtrate and the crude extract of Nymphaeaceae have a synergistic effect in anti-aging efficacy.

[0215] (2) MMP3 PCR

[0216] Matrix metallopeptidase 3 (MMP3) Primers:

[0217] F:5'-CTGGACTCCGACACTCTGGA-3'

[0218] R:5'-CAGGAAAGGTTCTGAAGTGACC-3'

[0219] MMP3 is a matrix metalloproteinase III that can degrade some protein components in the extracellular matrix, such as collagen. In the skin, the degradation of collagen is associated with skin aging. Some studies have shown that MMP3 may play a role in skin tissue remodeling, helping to improve skin elasticity and firmness by promoting the repair and regeneration of the extracellular matrix. In addition, MMP3 may be involved in regulating the proliferation and migration of skin cells. This is essential for the repair and regeneration process of the skin. After the test substance is applied to the cell model, it is tested by MMP3 Changes in gene expression are used to evaluate the anti-wrinkle efficacy of the test substance.

[0220] Table 20 Combinations of filtrate and crude extract of Hedysarum truncatum in different proportions MMP3 Table of relative mRNA levels of genes

[0221]

[0222] Conclusion: After UVA treatment, compared with BC, MMP3 The relative mRNA level of the gene increased significantly, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used. MMP3 The relative mRNA level of the gene decreased significantly, indicating that the positive control of the experiment was effective. The filtrate and the crude extract of the ground jellyfish were combined at a ratio of 100:1. MMP3 The gene expression levels of S BLISS >0, indicating that the filtrate and the crude extract of Nymphaeaceae have a synergistic anti-aging effect, indicating that the filtrate and the crude extract of Nymphaeaceae have a synergistic effect in anti-aging efficacy.

[0223] (3) COL1 PCR

[0224] Collagen I (COL1) Primers:

[0225] F:5'- GTGCGATGACTGATCTGTGA-3'

[0226] R:5'- CGGTGGTTTCTTGGTCGGT-3'

[0227] 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.

[0228] Table 21 Combinations of filtrate and crude extract of Hedysarum sphaerocephalum in different proportions COL1 Table of relative mRNA levels of genes

[0229]

[0230] Conclusion: After UVA treatment, compared with BC, COL1 The relative mRNA level of the gene decreased significantly, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used. COL1 The relative mRNA level of the gene increased significantly, indicating that the positive control of the experiment was effective. The filtrate and the crude extract of the ground jellyfish were combined at a ratio of 10:1. COL1 The gene expression levels of S BLISS >0, indicating that the filtrate and the crude extract of Nymphaeaceae have a synergistic anti-aging effect, indicating that the filtrate and the crude extract of Nymphaeaceae have a synergistic effect in anti-aging efficacy.

[0231] (4) COL3 PCR

[0232] Collagen III (COL3) Primers:

[0233] F: 5'-TTGAAGGAGGATGTTCCCATCT-3'

[0234] R: 5'-ACAGACACATATTTGGCATGGTT-3

[0235] 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.

[0236] Table 22 Combinations of filtrate and crude extract of Hedysarum truncatum in different proportions COL3 Table of relative mRNA levels of genes

[0237]

[0238] Conclusion: After UVA treatment, compared with BC, COL3 The relative mRNA level of the gene decreased significantly, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used. COL3 The relative mRNA level of the gene increased significantly, indicating that the positive control of the experiment was effective. The filtrate and the crude extract of the ground jellyfish were combined at a ratio of 1:10, 1:1 and 100:1. COL3 The gene expression levels of S BLISS >0, indicating that the filtrate and the crude extract of Nymphaeaceae have a synergistic anti-aging effect, indicating that the filtrate and the crude extract of Nymphaeaceae have a synergistic effect in anti-aging efficacy.

[0239] 4. Detection and analysis of anti-aging proteins (WB)

[0240] 4.1 Determination of the expression of anti-aging proteins

[0241] (1) HFF fibroblasts grown in 12-well plates were stimulated with UVA to establish the model.

[0242] (2) 24 hours after modeling, collect cells and add RIPA cell lysis buffer to extract cells.

[0243] 3) Protein quantification: Use a protein quantification method (such as the BCA method) to determine the extracted protein concentration.

[0244] (4) SDS-PAGE electrophoresis: Use polyacrylamide gel electrophoresis (SDS-PAGE) to separate protein samples according to molecular weight.

[0245] (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.

[0246] (6) Blocking: Place the membrane in 5% skimmed milk powder and incubate for 1 hour.

[0247] (7) Primary antibody incubation: Add the primary antibody (specific antibody) of the target protein to 5% skimmed milk powder and incubate overnight to allow it to fully bind to the target protein in the membrane.

[0248] (8) Washing: Wash the membrane with TBST buffer to remove unbound primary antibody.

[0249] 9) Secondary antibody incubation: Add the secondary antibody corresponding to the source of the primary antibody (e.g. goat anti-rabbit IgG) to the solution containing 5% skim milk powder and incubate for 2 hours 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.

[0250] (10) Washing: Wash the membrane with TBST buffer to remove unbound secondary antibody.

[0251] (11) Signal detection: Use the substrate chemiluminescence ECL method to detect the protein labeled with the secondary antibody, and use the ChemiDoc imaging system to image and save the target protein.

[0252] (12) Result analysis: ImageJ was used to perform quantitative analysis of the target protein based on the signal intensity and location.

[0253] 4.2 Anti-aging effect of filtrate + crude extract of Hedysarum truncatum (100:1) (WB)

[0254] Table 23 WB experiment design table

[0255]

[0256] The test results are shown in Table 24 and Fig.19 .

[0257] (1) MMP1 WB

[0258] MMP1 is a matrix metalloproteinase, which mainly decomposes interstitial collagen, including types I, II and III. Collagen is the main structural protein in the dermis of the skin. When collagen is degraded, the mechanical support of the skin is lost, which leads to the appearance of wrinkles. After the test object acts on the cell model, the anti-wrinkle effect of the test object is evaluated by detecting the changes in the expression of MMP1 protein.

[0259] Table 24 Relative expression values ​​of MMP1 protein in filtrate, crude extract of Hedyssa centifolia and different combinations

[0260]

[0261] Summary: After UVA treatment, the expression of MMP1 protein increased compared with BC, indicating that the anti-aging model was successfully established. Under UVA stimulation, the expression of MMP1 protein decreased significantly using the positive control TGF-β, indicating that the positive control of this experiment was effective. Under UVA stimulation, the expression of MMP1 protein in all monomers and compositions decreased significantly, indicating that all monomers and compositions have anti-aging effects. In addition, the expression level of MMP1 protein in the composition of filtrate and crude extract of Hedysarum sutchuenensis at a ratio of 100:1 was significantly decreased compared with that of the single filtrate and crude extract of Hedysarum sutchuenensis, and S BLISS >0, indicating that the filtrate and the crude extract of Nasturtium schrenkiana have a synergistic anti-aging effect. At the same time, compared with the combination of CLR bifid yeast and Nasturtium schrenkiana crude extract (100:1), the MMP1 protein expression level of the combination of filtrate and Nasturtium schrenkiana crude extract (100:1) showed a significant decrease, indicating that the combination of filtrate and Nasturtium schrenkiana crude extract has a higher inhibition rate on MMP1 protein than the combination of CLR bifid yeast and Nasturtium schrenkiana crude extract. In summary, the combination of filtrate and Nasturtium schrenkiana crude extract has a synergistic effect on anti-aging efficacy, and the effect is better than the anti-aging effect of the combination of CLR bifid yeast and Nasturtium schrenkiana crude extract.

[0262] 5. In vitro tyrosinase activity test to determine the whitening effect

[0263] 5.1 Determination of tyrosinase activity

[0264] (1) Preparation of stock solutions: 1) Tyrosinase solution: Prepared with pH 6.8 PBS to 100u / ml; 2) Levodopa solution: Prepared with pH 6.8 PBS to 1mg / ml; 3) Kojic acid (positive control): Diluted with pH 6.8 PBS to 0.3mg / ml.

[0265] (2) Add 100ul of tyrosinase solution to each sample tube, cover the lid, vortex and separate the sample tube and the background tube, and then incubate in a 37℃ water bath for 10 minutes.

[0266] (3) Add 400 μl of L-DOPA solution to each tube in turn, cover the tube, vortex and spin down, and control the reaction time of each tube to 5 minutes.

[0267] (4) Transfer the reaction solution in each tube into a 96-well plate and measure the absorbance at 475 nm.

[0268] (5) Calculate tyrosinase inhibition rate: Tyrosinase inhibition rate (%) = (1- )×100%.

[0269] (6) In the formula, T is the absorbance of the sample well, that is, the absorbance of the solution after the sample reacts with tyrosinase; T0 is the background absorbance of the sample; C is the average of three absorbances of the enzyme reaction tube, that is, the absorbance of the reaction between tyrosinase and DOPA when no sample is added; and C0 is the background absorbance of the solvent.

[0270] (7) 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 that the difference was statistically significant.

[0271] Table 25 Tyrosinase inhibition rate determination experimental sample solution table

[0272]

[0273] The test results are shown in Tables 26 to 28 and Figures 20 to 22 .

[0274] 5.2 Whitening effect of filtrate (in vitro tyrosinase activity)

[0275] Table 26 Numerical table of tyrosinase inhibition rate of filtrates of different concentrations

[0276]

[0277] Summary: The positive standard 0.3mg / ml kojic acid showed a good tyrosinase inhibition rate, indicating that the experimental system was successful. The filtrate with a concentration of 0.1mg / ml showed a good tyrosinase inhibition rate, indicating that the filtrate concentration of 0.1mg / ml has a good whitening effect.

[0278] 5.3 Whitening effect of crude extract of Rhizoma Cibotii (tyrosinase activity in vitro)

[0279] Table 27 The tyrosinase inhibition rate of crude extracts of different concentrations of Hedysarum truncatum

[0280]

[0281] Summary: The use of 0.3mg / ml kojic acid showed a good tyrosinase inhibition rate, indicating that the experimental system was successful. Different concentrations of the crude extract of the ground jellyfish showed a good tyrosinase inhibition rate, and it was concentration-dependent, indicating that the crude extract of the ground jellyfish has a good whitening effect.

[0282] 5.4 Whitening effect of different ratios of filtrate + crude extract of Hedysarum scabra (in vitro tyrosinase activity)

[0283] Table 28 The tyrosinase inhibition rate of different ratios of filtrate and crude extract of Hedysarum truncatum

[0284]

[0285] Conclusion: The positive standard 0.3mg / ml kojic acid showed a good tyrosinase inhibition rate, indicating that the experimental system was successful. When the ratio of filtrate: scutellaria baicalensis crude extract was 1:100, the tyrosinase inhibition rate increased significantly compared with the single filtrate and scutellaria baicalensis crude extract, and S BLISS >0, indicating that the filtrate and the crude extract of Herba Lycoris Radiatae have strong tyrosinase inhibition ability at the combination of 1:100, and have a synergistic whitening effect.

[0286] 6. Calculation of synergistic effects

[0287] Using the data from Tables 4, 7, 19-22, 24, and 28, the Bliss synergy score was calculated according to the Bliss independence model, with the formula: 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 29:

[0288] Table 29

[0289]

[0290] Note: The ratio refers to the mass ratio of the filtrate to the crude extract of Nymphaeaceae.

[0291] The above results show that the filtrate and the crude extract of Herba Lysimachiae have a synergistic whitening effect at a ratio of 1:100, and have a synergistic anti-aging effect at ratios of 100:1, 10:1, 1:10 and 1:1. It can be seen that the composition of the present invention has a good synergistic effect in multiple test items. Therefore, it can be determined that the combination of the two has a synergistic anti-aging and whitening effect.

[0292] 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, characterized in that It is composed of extract of Herba Lycopodii and fermented liquid of Bifidobacterium longum; The mass ratio of the Bifidobacterium longum fermentation liquid to the Herba Lycopodii extract is (1-100):1 or 1:(10-100); The preparation method of the Bifidobacterium longum fermentation liquid comprises: inoculating and culturing Bifidobacterium longum infantis subspecies to obtain seed liquid; fermenting the seed liquid, centrifuging it, taking the supernatant, and obtaining the fermentation filtrate of the Bifidobacterium longum infantis subspecies; The deposit number of the Bifidobacterium longum subspecies infantis is CGMCC NO.27851; The ground nasturtium extract is an alcohol extract of the ground nasturtium and its flower buds.

2. The composition according to claim 1, characterized in that The mass ratio of the Bifidobacterium longum fermentation liquid to the Herba Lycopodii extract is 100:1, 10:1, 1:1, 1:10 or 1:

100.

3. The composition according to claim 1, characterized in that The preparation method of the alcohol extract of the ground truncatum and its flower buds comprises: taking the ground truncatum and its flower buds, cold-immersing and extracting them with 75% ethanol for 3 times, each time for 24 hours, concentrating the extract, and obtaining a crude ground truncatum extract.

4. Use of the composition according to any one of claims 1 to 3 in the preparation of products with whitening and anti-aging effects.

5. The use according to claim 4, characterized in that: The anti-aging comprises at least one of the following I to IV:

1. Eliminate DPPH free radicals; II. Anti-UVA damage; III. Inhibition MMP1, MMP3 gene transcription, and / or inhibit the expression of MMP1 protein; IV. Promotion COL1 , COL3 Gene transcription; The whitening includes inhibiting the activity of tyrosinase.

6. Products with whitening and anti-aging effects, characterized in that: The invention comprises the composition according to any one of claims 1 to 3.

7. The product according to claim 6, characterized in that The concentration of the fermentation filtrate of Bifidobacterium longum is 0.02mg / ml-10mg / ml; the concentration of the extract of Herba Lycoris Radiatae is 0.02mg / ml-10mg / ml.

8. The product according to claim 6 or 7, characterized in that The products include cleansing cosmetics, care cosmetics, beauty / decorative cosmetics or medicines.

9. The product according to claim 8, characterized in that The dosage forms of the cleaning cosmetics, care cosmetics and beauty / decorative cosmetics include creams, emulsions, solutions, sprays, gels or powders; the dosage forms of the drugs include tablets, emulsions, granules, pills, liquid preparations, ointments or patches.

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