Composition for delaying skin aging and preparation method thereof

Abstract

The present invention provides a composition for delaying skin aging, comprising the following components: S-Rg2, S-Rh1, R-Rg2, R-Rh1, Rd, Rg6, Rg4, Rk3, Rh4, S-Rg3, R-Rg3, Rk1, Rg5, S-Rh2, R-Rh2, Rk2, and Rh3. The composition for delaying skin aging is prepared from raw materials of natural origin, can effectively inhibit cells from producing pro-inflammatory factors IL-1β, TNF-α, and IL-6, and effectively delay skin aging.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetic additives, and in particular to a composition for delaying skin aging and a preparation method thereof. Background Art

[0002] Skin aging is a normal physiological phenomenon that occurs with age. The main manifestations of skin aging include sagging skin and the formation of wrinkles.

[0003] As the body ages, blood circulation in the skin slows, and the subcutaneous fat layer begins to loosen and lose its elasticity. Facial lines from the earlobe to the chin may become sagging, and the skin above the cheekbones loses its firmness and firmness. Other signs of aging include wrinkles, such as fine lines at the corners of the eyes and crow's feet, and a downward shift of the highest point of the face, resulting in nasolabial folds, forehead wrinkles, and wrinkles at the neck. Aging also causes dryness on the face and body, and a sallow, dull complexion. Some people may also develop age spots or itchy skin. Besides natural aging, skin aging can also be accelerated by factors such as sunlight, mechanical forces, unhealthy lifestyle habits, and pollution.

[0004] As the saying goes, "Everyone has a desire for beauty." For thousands of years, people have been working hard and exploring ways to delay skin aging.

[0005] Scientists first proposed the concept of "inflamm-aging" in 2000, referring to the chronic pro-inflammatory state that occurs during the aging process. Clinical research has also revealed that inflamm-aging is a key determinant of the rate of aging and lifespan. While inflamm-aging is an inevitable and irreversible process of human aging, it does share common symptoms and can be measured using specific indicators. Therefore, interventions can, to a certain extent, prevent and delay the onset of this type of aging.

[0006] Our bodies harbor two types of "factors": pro-inflammatory factors (also known as inflammatory cytokines) and anti-inflammatory factors. These factors constantly compete in our bloodstream, and the outcome of this struggle not only determines the level of inflammation in our bodies but also directly influences the rate of aging. Simply put, "inflammatory aging" is caused by a deficiency of anti-inflammatory factors compared to pro-inflammatory factors. The main pro-inflammatory factors include TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β), and IL-6 (interleukin-6). The main anti-inflammatory factor is IL-10, a pleiotropic cytokine with important immunomodulatory functions.

[0007] Existing drugs that can inhibit pro-inflammatory factors, including non-natural inhibitors such as Enbrel, Pentoxifylline, and Sulfasalazine, are not suitable for use in areas such as skin care products.

[0008] How to obtain naturally derived products to delay skin aging and combat inflammatory aging is a question that our company's R&D team has been exploring. Summary of the Invention

[0009] In order to solve the above-mentioned problems existing in the prior art, the present invention provides a composition for delaying skin aging and a preparation method thereof, and achieves the following invention objectives: preparing a natural composition for delaying skin aging that effectively inhibits cells from producing IL-1β, IL-6, and TNF-α.

[0010] In order to achieve the above object of the invention, the technical solution adopted by the present invention is:

[0011] A composition for delaying skin aging comprises the following components: S-Rg2, S-Rh1, R-Rg2, R-Rh1, Rd, Rg6, Rg4, Rk3, Rh4, S-Rg3, R-Rg3, Rk1, Rg5, S-Rh2, R-Rh2, Rk2, and Rh3.

[0012] The distribution ratio of each component in the composition: The content ratio of each component in the composition is: S-Rg2: S-Rh1: R-Rg2: R-Rh1: Rd: Rg6: Rg4: Rk3: Rh4: S-Rg3: R-Rg3: Rk1: Rg5: S-Rh2: R-Rh2: Rk2: Rh3, the mass ratio is 5.1-6.1: 4.8-5.8: 2.8-4.2: 3.0-3.8: 1.0-1.5: 7.0-8.5: 12.5-15.5: 6.4-7.2: 13.5-15.0: 6.1-7.5: 3.4-3.8: 7.0-8.2: 10-12: 1.7-2.2: 1.0-1.2: 2.7-3.2: 3.4-3.8.

[0013] A method for preparing a composition for delaying skin aging comprises the following steps:

[0014] Step 1: Select the strain

[0015] The selected strain is Lactobacillus salpinax, which is commercially available and is a standard strain of the American ATCC, with the strain collection number ATCC15521.

[0016] Step 2: Vaccination

[0017] Under room temperature, Lactobacillus sackerii is inoculated into a basic culture medium; the basic culture medium is: MRS culture medium with a pH value of 4-6; and the inoculation amount is 1-1.5%.

[0018] Step 3: Fermentation

[0019] The inoculated basal culture medium is placed in an incubator for fermentation at a temperature of 37-40°C.

[0020] Step 4: Induction

[0021] After 48-72 hours of fermentation, add the inducer and ginsenosides to the basal medium. The inducer should be 0.1-10% of the total weight of the medium, and the ginsenosides should be 0.01-5% of the total weight of the medium. After addition, shake the medium thoroughly and continue fermentation for another 48-96 hours.

[0022] The raw material components of the inducer include ginseng, American ginseng, soybean and Panax notoginseng, and the mass ratio is 4-5:3-4:7-9:2-2.5.

[0023] The ginseng may also be ginseng tissue, tissue culture or adventitious root;

[0024] The American ginseng may also be the tissue, tissue culture or adventitious root of American ginseng;

[0025] The soybean may also be soybean tissue, tissue culture or adventitious root;

[0026] The Panax notoginseng can also be the tissue, tissue culture or adventitious root of Panax notoginseng.

[0027] The preparation method of the inducer:

[0028] 1. Raw material preparation: Prepare the raw materials according to the raw material ratio.

[0029] 2. Extraction: Grind ginseng, American ginseng, soybeans, and Panax notoginseng, then stir and mix to obtain a mixture. Add deionized water in an amount equal to four times the mass of the mixture. After adding the deionized water, microwave treatment is initiated. Heat and stir at 70-80°C, 200-300 rpm, for 8-10 minutes. After treatment, filter and retain the liquid phase to obtain an extract. Repeat this process three times. Microwave treatment: microwave power: 850-900W, microwave treatment time: 5 minutes.

[0030] 3. Extraction: Extract the extract three times with water-saturated n-butanol, with the amount of water-saturated n-butanol being 50-60% of the extract mass. After extraction, retain the n-butanol layer from each extraction and combine them to obtain the extract phase.

[0031] 4. Obtaining the inducer: Volatilize the n-butanol from the extract phase under reduced pressure to obtain the inducer.

[0032] Step 5: Post-fermentation treatment

[0033] After the fermentation is completed, the bacteria are first removed by filtration, and then the culture solution is concentrated to 5-20% of the volume of the culture solution to obtain a concentrated solution.

[0034] Add ethanol to the concentrate, shake for 2-3 minutes, centrifuge to remove insoluble matter, concentrate and evaporate to dryness to obtain a crude extract. The amount of ethanol added is 3-5 times the volume of the concentrate, and the ethanol concentration is 95-100%.

[0035] The crude extract was mixed with 95% ethanol at a mass ratio of 1:3, and after dissolution, 8-10 times of deionized water was added and mixed to obtain a dilution solution.

[0036] The diluted solution is purified by macroporous adsorption resin; after the saponin is adsorbed by the resin, it is washed with water to remove impurities; the saponin is eluted with alcohol with a concentration of more than 85%, and the eluate is evaporated to dryness to obtain a powder, which is the anti-skin aging composition. Beneficial effects

[0037] (1) The anti-skin aging composition of the present invention is prepared from raw materials of natural origin and is used as a skin care product or health care product for delaying skin aging. It is green and healthy. At the same time, it can effectively delay and improve inflammatory aging and is suitable for vigorous promotion.

[0038] (2) The anti-skin aging composition of the present invention can effectively inhibit the production of pro-inflammatory factors IL-1β, TNF-α, and IL-6 by cells, and significantly reduce the content of IL-1β, TNF-α, and IL-6; the efficacy is equivalent to that of SU.

[0039] (3) The anti-skin aging composition of the present invention can effectively promote the production of IL-10 by cells and significantly increase the content of IL-10 in serum; its efficacy is better than that of sulfasalazine. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Attachment Figure 1 The ELISA results for IL-1β content in each group in Example 3 are as follows;

[0041] Attachment Figure 2 The ELISA results for detecting TNF-α levels in each group in Example 3 are as follows;

[0042] Attachment Figure 3 The ELISA results for IL-6 content in each group in Example 3 are as follows;

[0043] Attachment Figure 4 The ELISA results for IL-10 content in each group in Example 3 are as follows;

[0044] Attachment Figure 5 This is the HPLC spectrum of the anti-skin aging composition of the present invention. Implementation Method

[0045] The following steps are involved:

[0046] 1. Raw material preparation

[0047] The raw material components of the inducer include ginseng, American ginseng, soybean and Panax notoginseng, with a mass ratio of 4.5:3:9:2.2.

[0048] Prepare the raw materials according to the raw material ratio.

[0049] 2. Extraction

[0050] Grind and stir ginseng, American ginseng, soybeans, and Panax notoginseng to form a mixture. Add deionized water at a rate of four times the mass of the mixture. After adding deionized water, microwave treatment was initiated. Then, heating and stirring were initiated at 78°C and 280 rpm for 10 minutes. After treatment, the liquid phase was filtered to obtain the extract. Repeat this process three times.

[0051] The microwave treatment was performed at a power of 880 W and a microwave treatment time of 5 min.

[0052] 3. Extraction

[0053] The extract was extracted three times with water-saturated n-butanol, with the amount of water-saturated n-butanol used being 55% of the mass of the extract. After extraction, the n-butanol layers from each extraction were retained and combined to obtain the extract phase.

[0054] 4. Obtain inducers

[0055] The n-butanol was evaporated from the extract phase under reduced pressure to obtain an inducer.

[0056] The following steps are involved:

[0057] Step 1: Select the strain

[0058] The selected strain is Lactobacillus salpinax, which is commercially available and is a standard strain of the American ATCC, with the strain collection number ATCC15521.

[0059] Step 2: Vaccination

[0060] At room temperature, Lactobacillus sackerii was inoculated into a basal culture medium; the basal culture medium was: MRS medium, pH value was 5; the inoculation amount was 1%.

[0061] Step 3: Fermentation

[0062] The inoculated basal culture medium was placed in an incubator for fermentation at a temperature of 38°C.

[0063] Step 4: Induction

[0064] After 48 hours of fermentation, the inducer prepared in Example 1 and ginsenosides were added to the basal medium. The inducer was added in an amount of 0.5% by weight of the total medium, and the ginsenosides were added in an amount of 1% by weight of the total medium. After adding the inducer, the culture was shaken and fermented for another 48 hours.

[0065] The raw material components of the inducer include ginseng, American ginseng, soybean and Panax notoginseng, with a mass ratio of 4.5:3:9:2.2.

[0066] Step 5: Post-fermentation treatment

[0067] After the fermentation is completed, the bacteria are first removed by filtration, and then the culture solution is concentrated to 10% of the volume of the culture solution to obtain a concentrated solution.

[0068] Ethanol was added to the concentrate, shaken for 3 minutes, centrifuged to remove insoluble matter, concentrated and evaporated to dryness to obtain a crude extract. The amount of ethanol added was 4 times the volume of the concentrate, and the ethanol concentration was 95%.

[0069] The crude extract was mixed with 95% ethanol at a mass ratio of 1:3, dissolved, and then 8 times of deionized water was added and mixed to obtain a dilution solution.

[0070] The diluted solution is purified by macroporous adsorption resin; after the saponin is adsorbed by the resin, it is washed with water to remove impurities; the saponin is eluted with 85% alcohol, and the eluate is evaporated to dryness to obtain a powder, which is the anti-skin aging composition.

[0071] After HPLC detection, the distribution ratio of the anti-aging functional additive components obtained was: S-Rg2:S-Rh1:R-Rg2:R-Rh1:Rd:Rg6:Rg4:Rk3:Rh4:S-Rg3:R-Rg3:Rk1:Rg5:S-Rh2:R-Rh2:Rk2:Rh3, and the mass ratio was 5.5:4.8:3.6:3.3:1.0:7.1:14.1:6.5:14.4:6.6:3.6:7.9:11.7:1.9:1.1:3.0:3.8.

[0072] The HPLC spectrum of the anti-skin aging composition test is as shown in the attached Figure 5 shown.

[0073] Detection method:

[0074] Step 1: Take RAW264.7 cells in the logarithmic growth phase and adjust the cell concentration to 1×10 5 / mL, 1 mL was added to each well of a 96-well plate, labeled as blank group, control group 1, control group 2, experimental group 1, and experimental group 2, with 6 replicates per group, and the process was repeated 3 times. The nutrient solution was RPMI1640 containing 10% fetal bovine serum.

[0075] Step 2: Place the marked well plate in a cell culture incubator at 37°C and 5% CO2 for overnight culture, and then replace each well with 1 mL of new nutrient solution.

[0076] The nutrient solution was RPMI1640 containing 10% fetal bovine serum.

[0077] Step 3: Intervention

[0078] Drugs were added to the wells of experimental group 1, experimental group 2, and control group 2, respectively, and intervention was performed for 2 hours; specifically:

[0079] Experimental Group 1: The anti-skin aging composition of the present invention (abbreviated as Rgs) prepared in Example 2 was added to the wells of the plate marked as Experimental Group 1, with a dosage of 5 mg / L per well.

[0080] Experimental group 2: Rgs was added to the wells marked as experimental group 2, with a dose of 10 mg / L per well.

[0081] Control group 2: Add the anti-inflammatory drug sulfasalazine (SU) to the wells marked as control group 2, with a dose of 10 mg / L per well.

[0082] Step 4: LPS induction

[0083] After intervention, LPS (endotoxin, i.e., lipopolysaccharide) was added to the wells marked as experimental group 1, experimental group 2, control group 1, and control group 2 for induction, with 100 ng / mL of LPS added to each well.

[0084] The blank group was treated with the same amount of PBS buffer. The plates were incubated for 8 h under the original culture conditions, and then the supernatants were collected and the levels of IL-1β, TNF-α, IL-6, and IL-10 in the supernatants were determined using ELISA kits according to the instructions.

[0085] The results of IL-1β content in the supernatant of each group are shown in the attached figure. Figure 1 As shown:

[0086] The results of TNF-α content in the supernatant of each group are shown in the attached figure. Figure 2 As shown:

[0087] The results of IL-6 content in the supernatant of each group are shown in the attached figure. Figure 3 As shown:

[0088] The results of IL-10 content in the supernatant of each group are shown in the attached figure. Figure 4 As shown:

[0089] Analysis Attachment Figure 1-3As can be seen, the IL-1β, TNF-α, and IL-6 levels in the control group 1 were significantly higher than those in the blank group, indicating that the IL-1β, TNF-α, and IL-6 levels in the serum were significantly increased after LPS induction. The IL-1β, TNF-α, and IL-6 levels in the experimental groups 1 and 2 were significantly lower than those in the control group 1, indicating that the intervention of the anti-skin aging composition of the present invention effectively reduced the levels of IL-1β, TNF-α, and IL-6 in the serum, indicating that the anti-skin aging composition of the present invention effectively inhibited the production of IL-1β, TNF-α, and IL-6 by cells. Among them, the effect of the experimental group 2 was basically the same as that of the control group 2, indicating that the efficacy of the anti-skin aging composition of the present invention in inhibiting the production of IL-1β, TNF-α, and IL-6 by cells is comparable to that of SU.

[0090] Analysis Attachment Figure 4 It can be seen that by comparing the blank group, control group 1, experimental group 1 and experimental group 2, the intervention of the anti-skin aging composition of the present invention effectively increased the IL-10 content in the serum, indicating that the anti-skin aging composition of the present invention effectively promotes the production of IL-10 by cells.

[0091] The IL-10 contents of experimental group 1 and experimental group 2 were higher than those of control group 2, among which the IL-10 content of experimental group 2 was significantly higher than that of other groups; this indicates that the efficacy of 5 mg / L dose of Rgs in promoting cell production of IL-10 is better than that of 10 mg / L dose of SU, and the efficacy of 10 mg / L dose of Rgs in promoting cell production of IL-10 is better.

[0092] The structural formulas of the components of the anti-skin aging composition of the present invention are:

[0093]

[0094] Unless otherwise specified, the ratios described in the present invention are all mass ratios, and the percentages described are all mass percentages.

[0095] Obviously, there are many specific implementation methods that can be changed under the concept of the present invention. Here, it should be stated that any changes made under the inventive concept of the present invention will fall within the scope of protection of the present invention.

Claims

1. A method for preparing a composition for delaying skin aging, characterized in that: The preparation method comprises the following steps: using Lactobacillus salquefetti as the strain for preparation, including the steps of inoculation, fermentation, induction, and post-fermentation treatment; the induction comprises fermentation culture for 48-72 hours, then adding inducer and ginsenoside to the basic culture medium, wherein the amount of the inducer added is 0.1-10% of the total mass of the culture medium, and the amount of ginsenoside is 0.01-5% of the total mass of the culture medium; the raw material components of the inducer include ginseng, American ginseng, soybean and Panax notoginseng, and the mass ratio is 4-5:3-4:7-9:2-2.5; A composition for delaying skin aging is prepared, and the content ratio of each component is: S-Rg2: S-Rh1: R-Rg2: R-Rh1: Rd: Rg6: Rg4: Rk3: Rh4: S-Rg3: R-Rg3: Rk1: Rg5: S-Rh2: R-Rh2: Rk2: Rh3, and the mass ratio is 5.1-6.1: 4.8-5.8: 2.8-4.2: 3.0-3.8: 1.0-1.5: 7.0-8.5: 12.5-15.5: 6.4-7.2: 13.5-15.0: 6.1-7.5: 3.4-3.8: 7.0-8.2: 10-12: 1.7-2.2: 1.0-1.2: 2.7-3.2: 3.4-3.

8.

2. The method for preparing a composition for delaying skin aging according to claim 1, characterized in that: The fermentation temperature is 37-40°C.

3. The method for preparing a composition for delaying skin aging according to claim 1, characterized in that: The ginseng is the tissue, tissue culture or adventitious root of ginseng; the American ginseng is the tissue, tissue culture or adventitious root of American ginseng; the soybean is the tissue, tissue culture or adventitious root of soybean; and the Panax notoginseng is the tissue, tissue culture or adventitious root of Panax notoginseng.

4. The method for preparing a composition for delaying skin aging according to claim 1, characterized in that: The preparation method of the inducer comprises extraction and extraction steps; the extraction comprises microwave treatment: the microwave power is 850-900W, and the microwave treatment time is 4-6min.

5. The method for preparing a composition for delaying skin aging according to claim 4, characterized in that: The extraction is performed by extracting with water-saturated n-butanol for 3-4 times.

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