A composition for improving mitochondrial activity and its use in cosmetics

Abstract

The application discloses a composition for improving mitochondrial activity and application thereof in cosmetics. The composition is prepared from a hyaluronic acid lysine copper chelate and an extract of European juniper berries. The composition can significantly promote the expression of NAD+, mitochondrial ATP and collagen III, and inhibit the expression of mitochondrial ROS, and has the effects of improving mitochondrial activity and effectively resisting aging, and can be used in cosmetics to improve skin relaxation and wrinkle problems.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and more specifically to a composition for enhancing mitochondrial activity and its application in cosmetics. Background Technology

[0002] The biological aging theory—the oxidative free radical theory—proposed by Harman in 1954 posits that the accumulation of oxidative cellular damage is a crucial factor in the aging process. Mitochondria, organelles that generate free radicals and are targets of oxidative stress, form the basis of this theory. Mitochondrial dysfunction and the production of reactive oxygen species (ROS) create a vicious cycle. Mitochondria are the primary site of oxidative phosphorylation and ATP synthesis within cells, providing energy for cellular activities. 95% of the energy required for cellular life activities comes from mitochondria, earning them the title of "cellular powerhouse." During aging, mitochondrial DNA mutations accumulate, oxidative phosphorylation levels decrease, and the expression of antioxidant enzymes becomes imbalanced, leading to excessive ROS production. This mitochondrial dysfunction and ROS production create a vicious cycle in the skin, generating a large number of free radicals. These free radicals oxidize the skin, damage collagen, and cause skin aging, sagging, loss of elasticity, and accelerated skin aging. Facial skin laxity is a common manifestation of skin aging and has become a hot research topic in cosmetic dermatology.

[0003] Currently, there are few cosmetics on the market that have a clear effect on enhancing mitochondrial activity. Therefore, there is a need to develop a cosmetic that effectively enhances mitochondrial activity to improve skin laxity and wrinkles. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a composition for enhancing mitochondrial activity and its application in cosmetics. The composition of this invention is a compound of hyaluronic acid-lysine-copper chelate and juniper fruit extract. Experimental verification has shown that this composition has excellent efficacy in enhancing mitochondrial activity and can be used in cosmetics to improve skin laxity and wrinkles, demonstrating promising application prospects in cosmetics.

[0005] To achieve the above-mentioned technical objectives, the first aspect of the present invention provides a composition for enhancing mitochondrial activity, characterized in that its components and mass fractions are: 0.05-1.0 parts of hyaluronic acid-lysine-copper chelate and 1.0-15.0 parts of Juniperus chinensis fruit extract.

[0006] The preferred ratio is: 0.3-0.8 parts of hyaluronic acid-lysine-copper chelate and 5.0-10.0 parts of juniper fruit extract.

[0007] Preparation method: Hyaluronic acid lysine copper chelate and juniper fruit extract are mixed together and dissolved in purified water (to make up to 100 parts).

[0008] The above-mentioned method for obtaining the Juniperus chinensis fruit extract involves commercially available products or the following method: Juniperus chinensis fruits are dried, pulverized, and passed through a 50-100 mesh sieve. The powder is then immersed in an ethanol solution (ethanol content 60%-85%) at a material-to-liquid ratio of 1:8-1:12 (g / mL) for 30-60 minutes. Ultrasonic extraction is then performed at a power of 150-250W for 30-50 minutes, repeated 2-3 times. The resulting filtrates are combined. The filtrate is concentrated under reduced pressure at 55-65℃ to obtain a Juniperus chinensis fruit concentrate. Butanediol (3-6 times its mass) is added to the concentrate, stirred thoroughly, and the insoluble substances are filtered out to obtain the Juniperus chinensis fruit extract. The concentration, based on crude drug, is 0.3-1.0 g / mL, meaning 0.3-1.0 g of dried Juniperus chinensis fruit powder yields 1 mL of Juniperus chinensis fruit extract (solution).

[0009] The hyaluronic acid-lysine-copper chelate used in this invention can be commercially available or obtained by salt formation (application number: CN202410478415.3). Specifically, sodium hyaluronate and lysine-copper chelate are mixed and dissolved evenly, then EDCI and NHS catalysts are added. After reacting at a controlled temperature, an appropriate amount of ethanol is added for precipitation and purification. The mixture is filtered, dehydrated with anhydrous ethanol, and dried under reduced pressure to obtain a blue granular powder.

[0010] A second aspect of this invention provides the use of the above-described composition in the preparation of cosmetics that enhance mitochondrial activity. Through experimental verification, this invention demonstrates that the above-described composition can promote the expression of NAD+ and mitochondrial ATP, thereby promoting the synthesis of type III collagen and inhibiting mitochondrial ROS expression, thus achieving an anti-aging effect.

[0011] Hyaluronic acid, lysine, and copper chelate have high moisturizing properties and better skin adhesion. They have a significant inhibitory effect on 5α-reductase, effectively controlling oil, preventing and treating acne vulgaris, improving the skin's water-oil balance, preventing seborrheic alopecia and improving hair quality, repairing skin and mucous membrane damage, promoting the production of elastin and collagen, preventing and reducing skin wrinkles, making the skin smooth and elastic, and achieving anti-wrinkle and firming effects, thus improving facial aging.

[0012] Juniper fruit has properties that promote diuresis, act as an antiseptic, relieve flatulence, and combat rheumatism. It also possesses antifungal activity, anti-inflammatory properties, helps prevent pigmentation, and enhances skin elasticity. Juniper fruit can also be taken orally to relieve indigestion, swelling, inflammation, and wounds.

[0013] A third aspect of the present invention provides an anti-aging skincare cosmetic comprising a composition that enhances mitochondrial activity.

[0014] Furthermore, based on the total mass of the anti-aging skincare cosmetic, the amount of the composition that enhances mitochondrial activity is 1.0-10.0%. More preferably, based on the total mass of the anti-aging skincare cosmetic, the amount of the composition that enhances mitochondrial activity is 3.0-8.0%.

[0015] Furthermore, the anti-aging skincare cosmetics also include one or more of the following: emulsifiers, emollients, moisturizers, thickeners, and preservatives.

[0016] Furthermore, by rationally adding the above-mentioned raw material components, this invention can also prepare different cosmetic formulations, such as essence water, essence liquid, facial mask, essence lotion, essence cream, etc. In addition, based on the above-mentioned basic cosmetic categories, further derivative cosmetic categories can be obtained, which obviously all fall within the protection scope of this application.

[0017] The beneficial effects of this invention are:

[0018] (1) The combination of sodium hyaluronate and lysine hydrochloride copper chelate in this invention can significantly promote NAD+ and mitochondrial ATP expression, promote Collagen III expression, and inhibit mitochondrial ROS expression, thereby achieving an effective anti-aging effect.

[0019] (2) Hyaluronic acid lysine copper chelate has a good effect on improving facial aging, but the cost of hyaluronic acid lysine copper chelate is high. In this invention, hyaluronic acid lysine copper chelate and juniper fruit extract are used in combination. When achieving similar or more significant effects on improving mitochondrial activity, the amount of hyaluronic acid lysine copper chelate is lower, which greatly saves costs.

[0020] (3) The preparation method of the anti-aging skin care cosmetic of the present invention is simple and easy to implement. Through human efficacy test, the essence cream with the above composition as the effective ingredient can significantly improve the skin elasticity R2, R5, R7, Q1 values, significantly reduce the F4 value, and significantly improve facial wrinkles, thus having a good anti-wrinkle effect. Detailed Implementation

[0021] The present invention will be further illustrated below by means of embodiments, but these embodiments are not intended to limit the invention to their scope. Based on the embodiments of the present invention, any modifications to the present invention made by those skilled in the art without inventive effort are within the scope of protection of the present invention. Furthermore, in the embodiments of the present invention, unless otherwise specified, all raw materials used in the preparation are commercially available products well-known to those skilled in the art.

[0022] Examples 1-4 and Comparative Examples 1-5:

[0023] The specific preparation method is shown in Table 1 below, calculated by mass parts. The preparation method is as follows: Hyaluronic acid-lysine-copper chelate and Juniperus chinensis fruit extract are mixed together and dissolved in purified water.

[0024] Table 1. List of components for specific embodiments and comparative examples.

[0025]

[0026] The extract of *Juniperus chinensis* fruit was obtained by the following method: *Juniperus chinensis* fruit was dried, pulverized, and passed through a 50-100 mesh sieve. The powder was then immersed in a 75% ethanol solution at a material-to-liquid ratio of 1:10 (g / mL) for 45 minutes. Ultrasonic extraction was then performed at a power of 200W for 40 minutes, repeated three times, and the resulting filtrates were combined. The filtrate was concentrated under reduced pressure at 55-65℃ to obtain a concentrated *Juniperus chinensis* fruit solution. Butanediol (4 times its mass) was added to the concentrated solution, and the mixture was stirred until homogeneous. Insoluble substances were filtered out to obtain the *Juniperus chinensis* fruit extract. The concentration was 0.5 g / mL (based on crude drug weight), meaning 0.5 g of dried *Juniperus chinensis* fruit powder yielded 1 mL of *Juniperus chinensis* fruit extract (solution).

[0027] The hyaluronic acid-lysine-copper chelate used in this invention embodiment was obtained through Example 1 of CN202410478415.3. Specifically, sodium hyaluronate and lysine-copper chelate were mixed and dissolved evenly, then EDCI and NHS catalysts were added. After reacting at a controlled temperature, an appropriate amount of ethanol was added for precipitation and purification. The mixture was filtered, dehydrated with anhydrous ethanol, and dried under reduced pressure to obtain a blue granular powder, samples 1-6.

[0028] Example 5: Anti-aging Essence Cream

[0029] The above-mentioned composition for enhancing mitochondrial activity was used to prepare an essence cream product containing this anti-aging composition, the formula of which is shown in Table 2.

[0030] Table 2. Formula of the Essence Cream

[0031]

[0032] Preparation method of essence lotion:

[0033] S1: Preparation of the aqueous phase: Add the raw materials from phase A separately, heat to 80-85℃ and stir to dissolve;

[0034] S2: Preparation of the oil phase: Add the raw materials from phase B separately and heat to 75-80℃ to melt;

[0035] S3: Combine phase A and phase B, turn on homogenizer (3000 rpm), homogenize for 12 minutes, and start cooling;

[0036] S4: When the temperature drops to 65-70℃, add phase C, stir to dissolve, and after dissolution, continue to cool down while adding phase D and stirring evenly.

[0037] S5: When the temperature drops to 40-45℃, add phase E, stir to dissolve and disperse evenly, and the essence cream product is obtained. Experimental Example 1: Experiment to promote NAD+ and mitochondrial ATP expression, promote Collagen III expression, and inhibit mitochondrial ROS expression.

[0038] Human skin fibroblasts (HSF) were used as the model cell line in this study. Fibroblasts were seeded into 24-well plates and incubated overnight in an incubator (37°C, 5% CO2). Working solutions of the test substances were prepared according to the test protocol (Table 3). When the cell deposition rate in the 24-well plates reached 40%–60%, the drugs were administered to the groups, with 2 mL of sample added to each well, and three replicates per group. The positive control group (PC1) received 2 mL of culture medium containing TGF-β1 per well; the positive control group (PC2) received 2 mL of culture medium containing vitamin E per well. After drug administration, the 24-well plates were incubated in an incubator (37°C, 5% CO2) for 24 hours. According to the test groups, except for the blank control group, all other groups were irradiated with UVA at a dose of 30 J / cm². 2 Irradiation time: 26 minutes.

[0039] After irradiation, the cells were incubated in a CO2 incubator (37℃, 5% CO2) for 24 hours. After incubation, the cells were washed twice with 1 mL / well of PBS, and 1 mL of RNAiso Plus was added to each well. After cell lysis by pipetting, the cells were collected. RNA was extracted, reverse transcribed into cDNA, and then detected by real-time PCR. -△△CT The method is used to calculate the results.

[0040] After irradiation, cells were incubated in a CO2 incubator (37℃, 5% CO2) for 24 hours, and then collected. Analysis was performed according to the ELISA kit instructions. Colorimetric assay: After incubation, cells were collected and analyzed according to the NADH kit instructions. Mitochondrial ATP assay: After incubation, cells were washed twice with 2 mL / well PBS, and 200 μL of lysis buffer was added to each well. Cells were lysed by pipetting and collected. ATP content was measured according to the kit instructions. Mitochondrial reactive oxygen species (ROS) assay: After irradiation, staining and fluorescence testing were performed according to the mitochondrial ROS staining kit instructions, followed by analysis.

[0041] GraphPad Prism was used for plotting, and the results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. A p-value of 0.01 < 0.05 was considered statistically significant, and a p-value of 0.01 < 0.01 was considered highly statistically significant.

[0042] Table 3 Test Plan

[0043]

[0044]

[0045] Note: Positive control 1 is a positive control for detecting Collagen III, and positive control 2 is a positive control for detecting NAD+, mitochondrial ATP, and mitochondrial ROS. Samples were diluted with water.

[0046] Table 4 Summary of Collagen III Content Detection Results

[0047] Sample Name Mean SD P-value Upward adjustment rate (vs NC) BC 1.00 0.07 / / NC 0.75 0.08 0.016# / PC 0.94 0.06 0.034* 25.33％ Example 1 0.92 0.05 0.035* 22.67％ Example 2 0.98 0.06 0.016* 30.67％ Example 3 1.12 0.07 0.004** 49.33％ Example 4 1.07 0.08 0.008** 42.67％ Comparative Example 1 0.90 0.05 0.04* 20.00％ Comparative Example 2 1.018 0.06 0.009** 35.73％ Comparative Example 3 1.026 0.07 0.01* 36.80％ Comparative Example 4 0.8 0.06 / —— Comparative Example 5 0.79 0.08 / ——

[0048] (Note: ## indicates p < 0.01 compared to the blank control; ** indicates p < 0.01 compared to the negative control; * indicates p < 0.01 compared to the negative control.) <p<0.05。)

[0049] Table 4 shows that the extract of Juniperus chinensis fruit did not promote CollagenIII gene expression. However, when hyaluronic acid-lysine-copper chelate was mixed with the extract of Juniperus chinensis fruit, it significantly promoted CollagenIII gene expression. This indicates that the two have a significant synergistic effect in promoting CollagenIII gene expression.

[0050] Table 5 Summary of NAD+ Concentration Detection Results (Unit Protein)

[0051]

[0052] (Note: ## indicates p < 0.01 compared to the blank control; ** indicates p < 0.01 compared to the negative control; * indicates p < 0.01 compared to the negative control.) <p<0.05。)

[0053] Table 5 shows that hyaluronic acid-lysine-copper chelate did not promote the concentration of NAD+ per unit protein. However, when hyaluronic acid-lysine-copper chelate was mixed with Juniperus chinensis fruit extract, it significantly promoted the increase of NAD+ concentration per unit protein. This indicates that the two have a significant synergistic effect in promoting the increase of NAD+ concentration per unit protein.

[0054] Table 6 Summary of Mitochondrial ATP Results

[0055]

[0056]

[0057] (Note: ## indicates p < 0.01 compared to the blank control; ** indicates p < 0.01 compared to the negative control; * indicates p < 0.01 compared to the negative control.) <p<0.05。)

[0058] Table 6 shows that Examples 1-4 and Comparative Examples 1-5 all significantly promoted the increase of mitochondrial ATP. It also demonstrates that the hyaluronic acid-lysine-copper chelate and the juniper fruit extract have a significant synergistic effect in promoting the increase of NAD+ concentration per unit protein.

[0059] Table 7 Summary of Mitochondrial ROS Analysis Results

[0060] Sample Name Relative IOD / average cell count SD P-value Inhibition rate (vs NC) BC 1.00 0.06 / / NC 1.88 0.11 0.000## / PC 1.06 0.08 0.000** 43.62％ Example 1 1.57 0.09 0.019* 16.49％ Example 2 1.3 0.12 0.004** 30.85％ Example 3 1.09 0.11 0.001** 42.02％ Example 4 1.23 0.09 0.001** 34.57％ Comparative Example 1 1.68 0.05 0.046* 10.64％ Comparative Example 2 1.55 0.08 0.014* 17.55％ Comparative Example 3 1.54 0.05 0.008** 18.09％ Comparative Example 4 1.68 0.07 0.046* 10.64％ Comparative Example 5 1.55 0.07 0.012* 17.55％

[0061] (Note: ## indicates p < 0.01 compared to the blank control; ** indicates p < 0.01 compared to the negative control; * indicates p < 0.01 compared to the negative control.) <p<0.05。)

[0062] As shown in Table 7, Examples 1-4 and Comparative Examples 1-5 all significantly inhibited mitochondrial ROS expression. It can also be seen that the hyaluronic acid-lysine-copper chelate and the juniper fruit extract have a significant synergistic inhibitory effect on mitochondrial ROS expression.

[0063] Experimental Example 2: Evaluation of Anti-aging Efficacy in Human Body

[0064] Example 5 was tested for its anti-aging efficacy on human skin. A total of 36 participants were tested, including 3 healthy Chinese men and 33 women, aged 34 to 55 years, who met the criteria for voluntary inclusion and exclusion.

[0065] Directions for use: After cleansing and applying toner in the morning and evening, apply an appropriate amount of cream evenly to the face and gently massage until absorbed. Use twice daily, once in the morning and once in the evening, for 28 consecutive days.

[0066] Testing time: before sample use (D0), before sample use (D14), 28 days after sample use (D28).

[0067] Test methods and test parameters:

[0068] 1. Skin elasticity tester The MPA580 is used to test skin elasticity. In this test, R2, R5, R7, and Q1 were used as parameters to evaluate skin elasticity, and F4 was used as a parameter to evaluate skin firmness. Higher R2, R5, R7, and Q1 values ​​indicate better skin elasticity. Lower F4 values ​​indicate firmer skin.

[0069] 2. The VISIA-CR facial imaging test quantifies the percentage of wrinkle area (forehead wrinkles, crow's feet, under-eye wrinkles) as a parameter to assess the improvement of facial wrinkles.

[0070] The test results are shown in Tables 8-13.

[0071] Table 8. Descriptive statistics of wrinkle level assessment (n=36)

[0072]

[0073]

[0074] Table 9. Descriptive statistics of skin elasticity R² (n=36)

[0075]

[0076] Statistical analysis results: "*": The difference is statistically significant (0.01≤P<0.05); "***": The difference is statistically significant (P<0.001).

[0077] Table 10. Descriptive statistics of skin elasticity R5 content (n=36)

[0078]

[0079] Statistical analysis results: "*": The difference is statistically significant (0.01≤P<0.05); "**": The difference is statistically significant (0.001≤P<0.01); "***": The difference is statistically significant (P<0.001).

[0080] Table 11 Descriptive statistics of skin elasticity R7 (n=36)

[0081]

[0082]

[0083] Statistical analysis results: "*": The difference is statistically significant (0.01≤P<0.05); "***": The difference is statistically significant (P<0.001).

[0084] Table 12 Descriptive statistics of skin elasticity Q1 (n=36)

[0085]

[0086] Statistical analysis results: "*": The difference is statistically significant (0.01≤P<0.05); "***": The difference is statistically significant (P<0.001).

[0087] Table 13 Descriptive statistics of skin firmness F4 (n=36)

[0088]

[0089] Statistical analysis results: "*": The difference is statistically significant (0.01≤P<0.05); "***": The difference is statistically significant (P<0.001).

[0090] As shown in Tables 8-13, during the 4-week test period, Example 4 significantly improved skin elasticity (R2, R5, R7, Q1 values), significantly reduced F4 value, and showed obvious improvement in facial wrinkles. Therefore, the essence cream containing this composition that enhances mitochondrial activity has good anti-wrinkle effects.

[0091] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A composition for enhancing mitochondrial activity, characterized in that, Its components and mass fractions are as follows: 0.05-1.0 parts of hyaluronic acid-lysine-copper chelate and 1.0-15.0 parts of Juniperus chinensis fruit extract; the method for obtaining the Juniperus chinensis fruit extract is as follows: Juniperus chinensis fruit is dried, pulverized and passed through a 50-100 mesh sieve, and then the powder is immersed in a 60%-85% ethanol solution and extracted by ultrasonic extraction; the obtained filtrate is concentrated under reduced pressure to obtain Juniperus chinensis fruit concentrate, and then 3-6 times the mass of butylene glycol is added to the Juniperus chinensis fruit concentrate, stirred evenly, and the insoluble substances are filtered out to obtain Juniperus chinensis fruit extract; the concentration is 0.3-1.0 g / mL based on crude drug.

2. The composition for enhancing mitochondrial activity as described in claim 1, characterized in that, Its components and mass fractions are as follows: 0.3-0.8 parts of hyaluronic acid-lysine-copper chelate and 5.0-10.0 parts of juniper fruit extract.

3. The composition for enhancing mitochondrial activity as described in claim 1, characterized in that, The hyaluronic acid lysine copper chelate is prepared by mixing and dissolving sodium hyaluronate and lysine copper chelate evenly, adding catalysts EDCI and NHS, reacting at a constant temperature, adding ethanol for precipitation and purification, filtering, dehydrating with anhydrous ethanol, and drying under reduced pressure to obtain a blue granular powder.

4. A composition for enhancing mitochondrial activity as described in any one of claims 1-3, characterized in that, Hyaluronic acid-lysine-copper chelate and juniper fruit extract were mixed together and dissolved in purified water to make up to 100 parts.

5. The use of the composition for enhancing mitochondrial activity according to any one of claims 1-3 in the preparation of cosmetics with anti-aging effects.

6. The application as described in claim 5, characterized in that, The composition achieves its anti-aging effect by promoting NAD+ and mitochondrial ATP expression, promoting type III collagen synthesis, and inhibiting mitochondrial ROS expression.

7. An anti-aging skincare cosmetic, characterized in that, The cosmetic product comprises the composition for enhancing mitochondrial activity as described in claim 4.

8. The anti-aging skincare cosmetic as described in claim 7, characterized in that, The amount of the composition for enhancing mitochondrial activity used is 1.0-10.0% based on the total mass of the anti-aging skincare cosmetic.

9. The anti-aging skincare cosmetic as described in claim 7, characterized in that, The anti-aging skincare cosmetics also include one or more of the following: emulsifiers, emollients, moisturizers, thickeners, and preservatives.

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