Efficient synergistic anti-aging composition and application thereof

Abstract

The invention provides an efficient synergistic anti-aging composition and application thereof, and belongs to the technical field of biological medicine preparations. According to the high-efficiency synergistic anti-aging composition provided by the invention, through scientific compatibility of specific active ingredients, a remarkable synergistic effect is generated, so that the high-efficiency synergistic anti-aging composition shows excellent comprehensive effects in the aspects of aging resistance and oxidation resistance. The composition is clear and stable in effect, and a core raw material solution is provided for developing efficient anti-aging skin care products. Meanwhile, according to the scheme, compounding is conducted on the basis of definite active ingredients, raw material obtaining is not strictly limited by the growth cycle, the region and the yield of specific plant resources, large-scale and standardized production can be achieved, dependence on direct extraction of rare plants is avoided, the raw material source is greatly widened, the stability of a supply chain is guaranteed, and the method is suitable for industrial production. The method has important industrial application value.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical formulation technology, and in particular to a highly efficient synergistic anti-aging composition and its application. Background Technology

[0002] With social development and improved living standards, consumers' demands for skincare products have shifted from basic cleansing and moisturizing to more targeted and effective skincare, with anti-aging remaining a core requirement. Skin aging is a complex physiological process, mainly manifested in wrinkles, sagging skin, decreased elasticity, and dull skin tone. Its internal mechanisms are closely related to multiple factors such as oxidative stress, collagen loss, and inflammatory responses. Therefore, the market's requirements for the efficacy of anti-aging skincare products are constantly increasing.

[0003] Currently, the sources of anti-aging active ingredients in the cosmetics industry are diverse, mainly including vitamins (such as vitamin C, vitamin E and their derivatives), peptides, retinol and its derivatives, natural plant extracts, and various fermentation products that have emerged in recent years. Among them, natural plant extracts are highly favored due to their wide availability and high consumer acceptance. Many plants, such as pomegranate, green tea, ginseng, and centella asiatica, have been proven to be rich in polyphenols, flavonoids, saponins, and other active substances, possessing potential for antioxidant, anti-inflammatory, and collagen-synthesizing promotion properties, and are widely used in anti-aging products.

[0004] In pursuit of more comprehensive anti-aging effects, the industry often employs a "compounding" strategy, combining active ingredients with different mechanisms of action to achieve synergistic or complementary effects. However, simple physical mixing does not always produce ideal results. Complex interactions may exist between different components, and improper compatibility can not only fail to achieve the synergistic effect of "1+1≥2," but may also lead to efficacy cancellation (antagonism) or reduced stability. Therefore, how to screen out combinations that can truly produce synergistic effects from numerous natural active substances and determine their optimal ratio is the key technology and challenge in developing highly effective compound anti-aging raw materials. In addition, some highly effective plant raw materials are limited by their rarity, slow growth, or complex extraction processes, making it difficult to meet the requirements of industrial production for cost, yield, and quality stability, which also restricts the development and promotion of related products. Summary of the Invention

[0005] The purpose of this invention is to provide a highly efficient synergistic anti-aging composition and its application, which solves the technical problems in the prior art where the efficacy of single plant anti-aging active substances is limited, simple compounding of different substances lacks synergy or even produces antagonistic effects, and the inability to stably supply high-quality anti-aging raw materials due to growth limitations and extraction difficulties of rare plant raw materials.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a synergistic anti-aging composition comprising cyanidin-3,5-O glucoside, kaempferol-rutin glycoside, and quercetin.

[0007] Preferably, the mass ratio of cyanidin-3,5-O glucoside, kaempferol-rutin glycoside, and quercetin is 0.9~1.5:0.1:1.4~2.0.

[0008] The present invention also provides the use of the above composition in the preparation of anti-aging cosmetics.

[0009] Preferably, the anti-aging cosmetic product can inhibit collagenase activity and / or promote type I collagen expression.

[0010] The present invention also provides the use of the above composition in the preparation of antioxidant cosmetics.

[0011] Preferably, the antioxidant cosmetic is able to scavenge DPPH free radicals and / or reduce the content of reactive oxygen species in cells.

[0012] The present invention also provides a cosmetic product containing the composition described herein.

[0013] Preferably, the total mass concentration of the composition in the cosmetic is from 0.001% to 10%.

[0014] Preferably, the cosmetic is a serum, lotion, emulsion, cream, or mask.

[0015] Preferably, the cosmetic also includes one or more cosmetically acceptable excipients selected from moisturizers, emulsifiers, thickeners, preservatives, fragrances, and pH adjusters.

[0016] The beneficial effects of this invention are: The highly efficient synergistic anti-aging composition provided by this invention, through the scientific formulation of specific active ingredients, produces a significant synergistic effect, exhibiting excellent comprehensive efficacy in anti-aging and anti-oxidation. This composition has a clear and stable efficacy, providing a core raw material solution for the development of highly effective anti-aging skincare products. Furthermore, this solution, based on the formulation of clearly defined active ingredients, is not strictly limited by the growth cycle, region, and yield of specific plant resources. Production can be scaled up and standardized, eliminating reliance on direct extraction from rare plants, greatly broadening the sources of raw materials and ensuring the stability of the supply chain, thus possessing significant industrial application value. Attached Figure Description

[0017] Figure 1 This is a graph showing the results of adjusting the fluorescence level of COL1 using different ratios of active ingredients according to the present invention; Figure 2The graph shows the results of adjusting ROS content using different proportions of active ingredient combinations according to the present invention. Detailed Implementation

[0018] This invention provides an anti-aging active composition with synergistic effects. The composition consists of three specific active ingredients: cyanidin-3,5-O-diglucoside, kaempferol-rutinoside, and quercetin.

[0019] Regarding cyanidin-3,5-O glucoside: It belongs to the anthocyanin class of compounds and is a water-soluble natural pigment widely found in dark-colored fruits, vegetables, and flowers (such as black roses, blueberries, and purple cabbage). Its chemical structure contains multiple phenolic hydroxyl groups, giving it significant antioxidant activity; it can scavenge free radicals and inhibit lipid peroxidation. In the cosmetics field, its known or potential uses include as an antioxidant, anti-inflammatory agent, and skin tone adjuster. This ingredient can be obtained through extraction, isolation, and purification from plants such as black roses, and is also commercially available.

[0020] Regarding kaempferol-rutin glycosides: These are flavonol glycosides, products of the combination of kaempferol and rutin. Kaempferol and its glycosides are found in various plants and possess antioxidant, anti-inflammatory, and antibacterial bioactivities. In skincare applications, their ability to scavenge free radicals and protect cells from UV damage is often a focus. This ingredient can also be obtained from plant materials such as black rose through extraction and chromatographic separation methods, or purchased from the market.

[0021] About quercetin: It is a common flavonol compound, abundant in many plants such as onions, apples, and tea. Quercetin is known for its powerful antioxidant, anti-inflammatory, and anti-allergic properties, and is often used in skincare products to soothe the skin, reduce oxidative stress damage, and aid in anti-aging. It can be obtained through plant extraction or chemical synthesis and is widely used in the food, pharmaceutical, and cosmetic industries.

[0022] This invention has discovered that combining the above three specific active ingredients can produce synergistic anti-aging and antioxidant effects that are superior to those of a single ingredient or other combinations.

[0023] In a preferred embodiment of the present invention, the mass ratio of the three active ingredients was optimized to obtain the best synergistic effect. The mass ratio of cyanidin-3,5-O glucoside, kaempferol-rutin glycoside, and quercetin is 0.9~1.5 : 0.1 : 1.4~2.0. More preferably, this mass ratio can be 1.0~1.4 : 0.1 : 1.5~1.9. More preferably, this mass ratio can be 1.1~1.3 : 0.1 : 1.6~1.8. Particularly preferred mass ratios are selected from 1:1:1, 1.5:0.1:1.4, or 0.9:0.1:2, as these specific ratios have shown particularly outstanding synergistic effects in experiments. The total content of the three active ingredients in the composition can be adjusted according to the dosage form and efficacy requirements of the final product. For example, in the concentrated raw materials of the composition, the total mass concentration can be 1% to 99%; when added to the final cosmetic, the total mass concentration is usually 0.001% to 10%, preferably 0.01% to 5%, more preferably 0.05% to 2%, and more preferably 0.1% to 1%.

[0024] This invention also provides the application of the above-described composition in the preparation of cosmetics with anti-aging effects. Specifically, the "anti-aging" effect can be manifested in the ability to inhibit the activity of collagenase, thereby slowing down collagen degradation; and / or to promote the expression and synthesis of type I collagen (COL1), thereby helping to improve skin elasticity and reduce wrinkles. The cosmetic, by including the composition of this invention, can achieve one or more of the above-described anti-aging biological effects.

[0025] This invention also provides the application of the above-described composition in the preparation of cosmetics with antioxidant effects. Specifically, the "antioxidant" effect can be manifested in the ability to effectively scavenge DPPH free radicals, demonstrating in vitro free radical scavenging capacity; and / or the ability to reduce the content of intracellular reactive oxygen species (ROS) in cell models, alleviating oxidative stress damage to skin cells. The cosmetics containing the composition of this invention can achieve one or more of the above-described antioxidant biological effects.

[0026] The present invention further provides a cosmetic containing the active composition described herein. In this cosmetic, the total mass concentration of the active composition has a wide range, from 0.001% to 10%. Preferred total mass concentration ranges include 0.005% to 5%, 0.01% to 3%, 0.05% to 2%, and 0.1% to 1%, depending on the product form and efficacy requirements. Most preferably, in some high-efficiency products, an addition of approximately 0.5% may be used.

[0027] The cosmetic product can be any dosage form commonly found in the art suitable for dermal administration. Preferably, it is a facial skin care product. Without limitation, the dosage forms of the cosmetic product include, but are not limited to: serums, toners, lotions, creams, masks (including sheet masks, clay masks, etc.), eye creams, skin oils, gels, etc. Each dosage form has its conventional matrix composition and physicochemical properties, and the compositions of the present invention are compatible with the conventional matrix of the corresponding dosage form.

[0028] In addition to the active composition of the present invention, the cosmetic typically also contains a variety of cosmetically acceptable excipients (or carriers, excipients) to form a complete, stable, and usable product. These excipients are well known to those skilled in the art and can be selected and combined according to the chosen dosage form. Exemplary excipients include, but are not limited to: Moisturizers, such as glycerin, propylene glycol, butylene glycol, sodium hyaluronate, sodium pyrrolidone carboxylate, amino acids, and sugar isomers, are used to maintain skin moisture.

[0029] Emulsifiers, such as fatty acid glycerides, polysorbates, cetearyl alcohol polyethers, lecithin, etc., are used to stabilize oil-water mixtures (emulsions, creams).

[0030] Thickeners, such as carbomer, xanthan gum, cellulose gum, and magnesium aluminum silicate, are used to adjust the viscosity and rheological properties of products.

[0031] Emollients / oils: such as squalane, jojoba oil, silicone oil, fatty alcohols, fatty acid esters, etc., are used to moisturize and soften the skin.

[0032] Preservatives, such as phenoxyethanol, p-hydroxyacetophenone, ethylhexylglycerin, and sodium benzoate, are used to prevent the growth of microorganisms and ensure product safety.

[0033] Fragrances: Edible or everyday flavorings added as needed to provide a pleasant aroma.

[0034] pH adjusters, such as citric acid, sodium citrate, lactic acid, and triethanolamine, are used to adjust the pH of the product to a skin-compatible range (e.g., pH 4.0-7.0, preferably 5.0-6.5).

[0035] Other active ingredients: Without affecting the efficacy of the composition of the present invention, other active ingredients may be added as needed, such as vitamin C and its derivatives, vitamin E, nicotinamide, peptides, plant extracts, etc.

[0036] The preparation method of the cosmetic can employ conventional processes in the art. It typically includes the following steps: heating the aqueous phase components (including water, water-soluble active ingredients, moisturizers, etc.) and oil phase components (including oils, emulsifiers, oil-soluble active ingredients, etc.) to a specific temperature (e.g., 60℃-85℃) to dissolve or melt uniformly; then, under stirring, adding the oil phase to the aqueous phase or vice versa, for emulsification and homogenization; after the system temperature drops to a suitable range (e.g., below 40℃), adding the composition of the present invention (if heat-sensitive, it should be added at a low temperature), fragrance, preservatives, etc., and stirring until uniform; finally, adjusting the pH to obtain the final product. For non-emulsified systems (such as serums and toners), the process is simpler, mainly involving dissolving each component sequentially in water and mixing uniformly.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1 This embodiment provides a combination of anti-aging active substances, which is obtained by combining cyanidin-3,5-O glucoside, kaempferol-rutin glycoside and quercetin in a 1:1:1 ratio.

[0039] Experimental Example 1 Based on Example 1, the following experiments were conducted: Raw material preparation Accurately weigh cyanidin-3,5-O-glucoside standard (A), quercetin standard (B), kaempferol-rutin glycoside standard (C), apigenin-7-O-glucoside standard (D), and phlorizin standard (E) with a purity >95%. Dissolve each in methanol to prepare a stock solution with a mass concentration of 100 μg / mL, and store at 4℃ protected from light for later use.

[0040] Constructing composite combinations Based on the above five raw materials, a compound system was constructed according to a preset ratio and screened in a unified in vitro evaluation system.

[0041] A total of 14 groups of samples to be tested and 1 group of positive controls were set up.

[0042] The final concentration of all samples in the test system was 100 μg / mL.

[0043] The group combinations are as follows: Group: Compound composition (mass ratio 1:1:1) 1: A+B+C 2: B+C+D 3: C+D+E 4: A+B+D 5: A+B+E 6: A+C+D 7: A+C+E 8: A+D+E 9: B+D+E 10:A 11:B 12:C 13:D 14:E Positive control group: VC Screening for combinations of active ingredients with synergistic anti-aging effects: Collagenase inhibition rate detection Solution preparation: Dilute each group of samples with DMEM medium to 100 μg / mL, consistent with the concentration of the positive control.

[0044] Enzyme reaction system: Collagenase working solution was prepared at 0.1 U / mL and preheated at 37℃ for 10 min; FALGPA substrate working solution was prepared at 1 mmol / L and stored in the dark; buffer, sample / control solution and enzyme working solution were added in sequence and incubated at 37℃ for 15 min; then substrate was added and incubated at 37℃ for 60 min; the reaction was terminated by adding a terminator.

[0045] Detection: Place the microplate into the microplate reader, set the detection wavelength to 405nm, subtract the absorbance of the blank control group, and read the absorbance value (OD value) of each well in each group and record the data.

[0046] Calculation: Collagenase inhibition rate = (sample well OD - zeroing well OD) / (solvent control well OD - zeroing well OD) * 100% The results are shown in Table 1.

[0047] Table 1 Results of regulating collagenase inhibition rate

[0048] The experimental results showed that among the first 9 groups of samples, A+B+C=1:1:1, the DPPH scavenging rate was the highest, indicating that there may be a synergistic effect among the active ingredients cyanidin-3,5-O glucoside, kaempferol-rutin glycoside, and quercetin.

[0049] This indicates that the active ingredient combination (cyanidin-3,5-O glucoside, kaempferol-rutin glycoside, quercetin) may reduce the loss of type I collagen and decreased skin elasticity caused by aging by inhibiting collagenase, thus delaying the skin aging process.

[0050] Antioxidant efficacy test Screening for synergistic combinations with antioxidant effects: Reagent and Sample Preparation DPPH ethanol solution: Dilute 1,1-diphenyl-2-picrylhydrazine / DPPH to a final volume with ethanol to prepare a 0.04 mg / mL DPPH ethanol solution, and store in the dark.

[0051] Sample preparation: Dilute each group of samples with methanol to 100 μg / mL, consistent with the concentration of the positive control.

[0052] Testing process Experimental group: Take 20 μL of sample into a deep well plate, add 180 μL of DPPH ethanol solution, mix well, place in the dark for 30 min, shake continuously, and measure the absorbance at 520 nm.

[0053] Blank group: Take the same volume of sample solution as the experimental group and add 180 μL of ethanol solution. Mix well and place in the dark for 30 min with continuous shaking, and measure the absorbance at 520 nm.

[0054] Control group: 20 μL of distilled water was added to 180 μL of DPPH ethanol solution. After mixing, the mixture was placed in the dark for 30 min with continuous shaking, and the absorbance at 520 nm was measured.

[0055] Positive control group: Take 20 μL of Vc solution (2.2) into a test tube and add 180 μL of DPPH ethanol solution. Mix well and place in the dark for 30 min with continuous shaking, and measure the absorbance at 520 nm.

[0056] calculate: DPPH removal rate (%) = A0 - A1 + A2 / A0 * 100% Where A0 is the absorbance value of the control group; A1 is the absorbance value of the experimental group; and A2 is the absorbance value of the blank group. The results are shown in Table 2 below: Table 2 DPPH removal rate results

[0057] Experimental results showed that the A+B+C group had the highest DPPH scavenging rate among the nine sample groups (1:1:1), indicating that the combination of active ingredients cyanidin-3,5-O glucoside, kaempferol-rutin glycoside, and quercetin had the best effect. Furthermore, the DPPH free radical scavenging rate of the A+B+C sample group was significantly higher than that of each individual group and other formulation groups, indicating that there is a synergistic effect among the specific three ingredients provided by this invention.

[0058] Experiment Example 2 To verify the synergistic effect of the three active ingredients and to further determine the optimal ratio of the three active ingredients, the following sample groups and one positive control group were set up: Table 3. Sample group settings with different proportions

[0059] ROS content test 1) Cell seeding: at 8 × 10 4 Seed cells at a density of cells / well into 24-well plates and incubate overnight in an incubator (37°C, 5% CO2).

[0060] 2) Solution preparation: Prepare 100 μg concentration of sample working solution according to the sample setup scheme in Table 3.

[0061] 3) Radiation: Untreated cells were used as blank controls, and the remaining groups were stimulated with UVA according to the groups in Table 3, with the VC+VE group as a positive control.

[0062] 4) Sample loading: Add the drug according to Table 3 and continue culturing for 24 h; 5) ROS content detection: The DCFH-DA probe stock solution was diluted with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μmol / L. 500 μL of the diluted DCFH-DA probe was added to each well and incubated in a cell culture incubator at 37℃. After 30 min, the cells were washed four times with serum-free DMEM culture medium to thoroughly remove any probe that had not entered the cells. The cells were observed and photographed using a fluorescence microscope at an excitation wavelength of 488 nm.

[0063] The results are as follows Figure 1 As shown, the fluorescence results are as follows: Figure 2 As shown in Table 4: Table 4. Results of ROS content test

[0064] Based on a UVA-stimulated human fibroblast model, samples A+B+C=1:1:1 and A+B+C=1.5:0.1:1.4 showed significantly lower ROS levels at a concentration of 100% μg / mL, with statistically significant differences compared to the NC group. p <0.05); A+B+C=0.9:0.1:2 At a concentration of 100 μg / mL, the ROS content was significantly reduced, and the difference was statistically significant compared with the NC group ( p <0.01). This indicates that samples A+B+C=1:1:1, A+B+C=1.5:0.1:1.4, and A+B+C=0.9:0.1:2 have antioxidant capabilities.

[0065] Collagen I content test The above seven combinations of active ingredients were used to test the samples, and the method is as follows: 1. Prepare test samples The seven active ingredient combinations were prepared and diluted to a concentration of 100 μg / mL using DMEM culture medium, and the samples were then labeled and grouped.

[0066] 2. Detection of COL1 fluorescence Irradiation: Irradiation was carried out when the cell deposition rate in the 24-well plates reached 40%–60%. The negative control group, positive control group, and sample group received irradiation. The dose was 9 J / cm. 2 The system was exposed to UVA radiation, while a control group was placed in the same environment (UVA radiation dose of 0 J / cm²). 2 ).

[0067] Sample loading: After irradiation, sample loading is performed. 1 mL of cell culture medium is added to each well of the blank control group and negative control group; the positive control group... Add 1 mL of culture medium containing 100 μg / mL VC and 7 μg / mL VE to each well; add 1 mL of the corresponding concentration to each well of the sample group. The culture medium of the test substance.

[0068] Immunofluorescence assay for Collagen I expression: Cell collection: After sample loading, discard the supernatant and wash the cells three times with PBS. Immunofluorescence staining: Perform the routine immunofluorescence staining procedure. The main steps are: fixation, blocking, addition of primary antibody, addition of secondary antibody, counterstaining with DAPI, and then taking pictures using a fluorescence microscope.

[0069] Results analysis: Image Pro Plus software was used to quantitatively analyze the fluorescence intensity and expression level of Collagen I.

[0070] The data results are shown in Table 5: Table 5. Results of Collagen I content test

[0071] Compared with the BC group, the human fibroblasts in the NC group received a dose of 9 J / cm². 2 After UVA radiation, Collagen I expression decreased significantly by 33%. p <0.01) indicates that the modeling was successful.

[0072] Compared with the NC group, the PC group (VC+VE) significantly increased the expression level of Collagen I by 48.5%. p<0.01), indicating that the positive control test was effective.

[0073] Compared with the NC group, in sample A+B+C=1:1:1 at a concentration of 100% (V / V), the expression level of Collagen I in human fibroblasts was significantly increased by 24.2% ( p <0.05); Samples A+B+C=1.5:0.1:1.4 and A+B+C=0.9:0.1:2 showed significantly increased Collagen I expression in human fibroblasts at a concentration of 100% (V / V) by 81.8% and 78.8% (V / V, respectively). p <0.01).

[0074] Based on a UVA-stimulated human fibroblast model, samples A+B+C=1:1:1, A+B+C=1.5:0.1:1.4, and A+B+C=0.9:0.1:2, at a concentration of 100% (V / V), showed significantly increased Collagen I expression in human fibroblasts, with statistically significant differences compared to the NC group. p The value <0.05 indicates that samples A+B+C=1:1:1, A+B+C=1.5:0.1:1.4, and A+B+C=0.9:0.1:2 have anti-aging effects.

[0075] As shown in the above embodiments, this invention provides a highly efficient synergistic anti-aging composition composed of cyanidin-3,5-O glucoside, kaempferol-rutin glycoside, and quercetin, and its application. Experiments show that this specific combination significantly outperforms individual components and other test combinations in the evaluation of anti-aging and antioxidant effects, confirming a clear synergistic effect among the three. Further ratio optimization experiments determined that the synergistic effect is most pronounced when the three are combined in a specific mass ratio. This composition exhibits excellent ability to inhibit collagen degradation, promote collagen production, scavenge free radicals, and reduce oxidative damage in cell models. Therefore, the composition of this invention is a highly efficient and stable anti-aging active ingredient that can be used to prepare cosmetics with anti-aging and antioxidant effects.

[0076] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A synergistic anti-aging composition characterized in that, consisting of cyanidin-3,5-O-glucoside, kaempferol-rutinoside and quercetin.

2. The composition of claim 1, wherein, The mass ratio of the cyanidin-3,5-O-glucoside, kaempferol-rutinoside and quercetin is 0.9-1.5:0.1:1.4-2.

0.

3. Use of the composition of claim 1 or 2 in the preparation of an anti-aging cosmetic.

4. Use according to claim 3, characterized in that, The anti-aging cosmetic is capable of inhibiting collagenase activity and / or promoting type I collagen expression.

5. Use of the composition of claim 1 or 2 in the preparation of an antioxidant cosmetic.

6. Use according to claim 5, characterized in that, The antioxidant cosmetic is capable of scavenging DPPH free radicals and / or reducing intracellular reactive oxygen species content.

7. A cosmetic product, characterized by containing the composition of claim 1 or 2.

8. The cosmetic product according to claim 7, characterized in that, The total mass concentration of the composition in the cosmetic is 0.001% to 10%.

9. The cosmetic product according to claim 7, characterized in that, The cosmetic is a serum, a lotion, an emulsion, a cream or a mask.

10. The cosmetic according to any one of claims 7 to 9, characterized in that, The cosmetic further comprises one or more cosmetically acceptable adjuvants selected from a humectant, an emulsifier, a thickening agent, a preservative, a fragrance, a pH adjusting agent.

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