Anti-aging composition as well as preparation method, skin care product and application thereof

Abstract

The invention provides an anti-aging composition as well as a preparation method, a skin care product and application thereof. The anti-aging composition is prepared from a C6-C3-C6 skeleton compound, cationic amino acid and beta-alanyl hydroxyprolyl diaminobutyric acid benzylamine. The C6-C3-C6 structural compound is natural plant extracts such as quercetin, baicalin and the like, and has excellent oxidation resistance and high safety. Cell and in-vitro experiments prove that the combined action of the three components can effectively reduce the generation of reactive oxygen species (ROS) and enhance the anti-inflammatory action, thereby delaying the cell senescence process. On the basis, carbon dioxide is injected into the anti-aging composition, so that the anti-oxidation and anti-inflammatory capacities of the anti-aging composition are further improved. In addition, the preparation process of the anti-aging composition is simple, the safety and the anti-aging performance of the anti-aging composition are evaluated through various cell experiments and human body trials, and the anti-aging composition is suitable for human skin and can be widely added into various skin care products such as face cream, emulsion and essence.

Description

Technical Field

[0001] The present invention belongs to the technical field of daily chemicals, and in particular relates to an anti-aging composition, a preparation method thereof, a skin care product and applications thereof. Background Art

[0002] Oxidative stress and cellular inflammation are two key factors in the aging process, interacting in complex ways that collectively accelerate the aging process. Oxidative stress stems from an imbalance between the production and clearance of reactive oxygen species (ROS), which damages key cellular components such as lipids, proteins, and DNA. Excessive accumulation of ROS triggers a cellular stress response, leading to cell cycle arrest and thus promoting cellular senescence. Concurrently, cellular inflammation is a response of the immune system to injury and infection, and long-term, chronic, low-grade inflammation is closely associated with aging. Senescent cells release a large number of proinflammatory cytokines, forming the senescence-associated secretory phenotype (SASP). This phenotype not only exacerbates chronic inflammation but also drives normal cells toward a senescent state. Notably, ROS can activate inflammatory signaling pathways (such as NF-κB), promoting the expression and release of inflammatory factors. The inflammatory response, in turn, increases ROS production, creating a vicious cycle that further accelerates cellular and tissue aging.

[0003] Anti-aging intervention strategies usually adopt two main approaches: one is to neutralize ROS through antioxidants and reduce oxidative damage, that is, antioxidant therapy; the other is to inhibit chronic inflammatory responses through anti-inflammatory drugs and reduce the impact of SASP, that is, anti-inflammatory therapy.

[0004] In current anti-aging research, combined anti-aging technologies have become a mainstream trend, primarily including the combined use of antioxidants and anti-inflammatory drugs, therapies to eliminate senescent cells (such as senolytic drugs), and stem cell therapy. While these approaches theoretically hold promise for slowing the aging process and treating age-related diseases, they face several potential drawbacks in practical applications:

[0005] 1. Although antioxidants can effectively neutralize ROS and reduce oxidative damage, excessive intake may disrupt normal redox signals in cells, thereby affecting the physiological functions of cells;

[0006] 2. Anti-inflammatory drugs are mostly chemically synthesized, and their biosafety may be inferior to that of natural ingredients. Long-term use may suppress the normal response of the immune system, increase the risk of infection, and may cause more adverse reactions;

[0007] 3. Although stem cell therapy has the potential to repair damaged tissues, the reliability of stem cell sources, quality standards and potential carcinogenic risks are still issues that need to be addressed.

[0008] In addition, many of today's newly developed combined anti-aging technologies have not yet undergone long-term clinical testing, and their long-term efficacy and safety still need to be fully tested through extensive large-scale clinical trials.

[0009] Although combined anti-aging technology has great potential in theory, many difficult problems must be solved in actual application, such as treatment specificity, potential adverse reactions, complex ingredients, and biosafety and effectiveness. Summary of the Invention

[0010] To address the problems of overdose, safety concerns, and insufficient validation in existing combined anti-aging technologies, the present invention provides an anti-aging composition, its preparation method, skin care products, and applications. The anti-aging composition contains three active ingredients: a C6-C3-C6 backbone complex, cationic amino acids, and β-alanylhydroxyprolyldiaminobutyric acid benzylamine. The C6-C3-C6 backbone complex is derived from natural plant extracts such as quercetin and baicalin. These ingredients possess antioxidant properties and are highly safe. Cell-based and in vitro experiments have demonstrated that the synergistic effect of the three active ingredients can significantly reduce reactive oxygen species (ROS) production, enhance anti-inflammatory effects, and delay cell aging. Furthermore, the addition of carbon dioxide to the anti-aging composition can further enhance its antioxidant and anti-inflammatory effects. The composition is simple to prepare, its safety has been tested through cytotoxicity testing, and its anti-aging properties have been validated through multiple tests (such as CAA and ELISA) and human efficacy evaluations, making it suitable for application to human skin. It can be widely incorporated into skin care products such as creams, lotions, and serums to meet the needs of different skin types, providing consumers with a comprehensive anti-aging skin care solution.

[0011] In order to achieve the above object, the present invention adopts the following technical solutions:

[0012] In a first aspect, an anti-aging freeze-dried powder composition is provided, comprising the following components in parts by weight: 10.0% to 75.0% of a C6-C3-C6 skeleton complex; 7.0% to 80.0% of a cationic amino acid; and 10.0% to 65.0% of benzylamine β-alanylhydroxyprolyldiaminobutyrate. In a human efficacy evaluation experiment, the freeze-dried powder was compounded with a solubilizing agent, a polyol, and the like to form an anti-aging composition essence.

[0013] In some embodiments, the C6-C3-C6 skeleton complex is one or more of quercetin, baicalin, myricetin, and luteolin; and / or, the mass fraction of the C6-C3-C6 skeleton complex is 10.0% to 75.0%; and / or, the mass fraction of β-alanylhydroxyprolyldiaminobutyric acid benzylamine is 10.0% to 65.0%; and / or, the cationic amino acid is one or more of arginine, polyarginine, lysine, and histidine; and / or, the mass fraction of the cationic amino acid is 7.0% to 80.0%.

[0014] In a second aspect, an anti-aging essence composition is provided, which is in the form of an essence; the composition contains, by weight percentage of the total mass: 0.05% to 0.75% of the above-mentioned anti-aging freeze-dried powder composition; 5% to 10% of a dissolution agent; 4% to 10% of a polyol; 5% to 10% of oil; and the remainder is deionized water and a preservative.

[0015] In some embodiments, the solubilizing agent is one or more of sorbitan monooleate polyoxyethylene ether, glycerol polyether-26, Pluronic F127, and lecithin; and / or, the polyol is one or more of butylene glycol, propylene glycol, pentylene glycol, hexylene glycol, and ethoxydiglycol.

[0016] In some embodiments, the mass fraction of the solubilizing agent is 5.15%; and / or the mass fraction of the polyol is 4.50%; and / or the mass fraction of the oil is 5.00%.

[0017] In some embodiments, the composition further comprises: CO2, wherein the CO2 is in a saturated state within the anti-aging essence composition.

[0018] In a third aspect, a method for preparing an anti-aging freeze-dried powder composition is further provided, comprising the steps of:

[0019] The C6-C3-C6 skeleton complex, cationic amino acids and β-alanylhydroxyprolyldiaminobutyric acid benzylamine are fully dissolved in deionized water by ultrasonic method according to the weight ratio, and the operating temperature is 60-80°C. The mixed solution is then placed in liquid ammonia and rapidly cooled and frozen. Finally, the anti-aging freeze-dried powder composition is obtained by freeze-drying.

[0020] In a fourth aspect, a method for preparing an anti-aging essence composition is further provided, comprising the steps of:

[0021] The anti-aging freeze-dried powder composition according to claim 1 or 2 is fully dissolved in a solubilizing agent and a polyol by ultrasonic method according to a weight ratio, and the operating temperature is 60-80°C. Then, oil is added, stirred uniformly, added to deionized water, stirred to cool, and a preservative is added to obtain the anti-aging essence composition.

[0022] In some embodiments, the anti-aging serum composition is charged with CO2 until saturated.

[0023] In a fifth aspect, an application of an anti-aging composition in skin care products is provided, wherein the anti-aging composition is the above-mentioned anti-aging freeze-dried powder composition or the above-mentioned anti-aging essence composition.

[0024] In a sixth aspect, a skin care product is also provided, comprising the above-mentioned anti-aging composition, or the anti-aging composition prepared by the above-mentioned preparation method.

[0025] Compared with the existing technology, the anti-aging composition and its preparation method, skin care product and application provided by the present invention have the following beneficial effects:

[0026] 1. The anti-aging composition provided by the present invention adopts three active ingredients: C6-C3-C6 skeleton complex, cationic amino acids and β-alanylhydroxyprolyldiaminobutyric acid benzylamine. The C6-C3-C6 skeleton complex is derived from natural plant essences, including quercetin, baicalin, myricetin, luteolin, etc. These ingredients have antioxidant properties, are highly safe, and are mild and harmless to the human body. Through a series of rigorous cellular and in-depth studies at the in vitro level, it was found that when these three active ingredients are used in combination in a specific proportion, they can exert a synergistic effect, show more excellent antioxidant properties, effectively reduce the generation of reactive oxygen species (ROS), and thus protect cells from oxidative damage. In addition, the anti-inflammatory effect of the composition has also been significantly enhanced, which helps to relieve skin inflammation and maintain skin health. It has made breakthrough progress in the field of anti-aging and significantly delayed cell aging.

[0027] 2. The anti-aging composition provided by this invention has undergone further technical processing, namely, the addition of carbon dioxide into the anti-aging composition system. Carbon dioxide can effectively dissolve in the anti-aging composition, improving the solubility and bioavailability of the active ingredients in the composition. Thanks to this optimization, the absorption rate of the active ingredients is greatly improved, allowing them to more efficiently penetrate the skin barrier and penetrate deep into the skin's underlying layers. The active ingredients can fully exert their antioxidant and anti-inflammatory effects within the skin, effectively defending against free radical damage, reducing inflammatory responses, and further enhancing the overall efficacy of the anti-aging composition.

[0028] 3. The preparation method of the anti-aging composition provided by the present invention is simple, and its safety has been verified by cytotoxicity testing. The anti-aging performance of the composition has been confirmed by multiple testing methods such as intracellular antioxidant test (CAA), enzyme-linked immunosorbent assay (ELISA), real-time fluorescence quantitative PCR (RT-qPCR) technology, and β-galactosidase activity detection. At the same time, through human efficacy evaluation experiments, the efficacy of the anti-aging composition of the present invention and the anti-aging composition compounded with CO2 in delaying skin aging was demonstrated. Based on these positive evaluation results, the anti-aging composition provided by the present invention has been confirmed to be suitable for application to human skin, in order to bring a significant anti-aging skin care experience to consumers;

[0029] 4. The anti-aging composition provided by this invention has extremely high compatibility and flexibility, and can be widely incorporated into various skin care products, such as creams, lotions, and serums. Its formulation ensures stable activity in various skin care matrices, effectively enhancing the anti-aging efficacy of these products, meeting the needs of different skin types and providing consumers with a comprehensive skin care solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention will be further described below with reference to the accompanying drawings and examples.

[0031] Figure 1 Graph showing the cytotoxicity test results of different anti-aging compositions in Example 8;

[0032] Figure 2 Graph showing the test results of the intracellular antioxidant capacity of different anti-aging compositions in Example 9;

[0033] Figure 3 This is a graph showing the test results of the anti-aging composition and the anti-aging composition@CO2 on the intracellular anti-inflammatory ability;

[0034] Figure 4 This is a graph showing the test results of the anti-aging composition and the anti-aging composition@CO2 on the expression level of ICAM mRNA in cells;

[0035] Figure 5 This is a graph showing the test results of the anti-aging composition and the anti-aging composition@CO2 on the expression level of VCAM mRNA in cells;

[0036] Figure 6 This is a graph showing the test results of the anti-aging composition and the anti-aging composition@CO2 on the expression level of cellular MMP-1 mRNA;

[0037] Figure 7 This is a test result diagram of the effect of the anti-aging essence composition on the skin elasticity parameter (R2);

[0038] Figure 8 The second test result graph shows the effect of the anti-aging essence composition on the skin elasticity parameter (R2);

[0039] Figure 9 The third test result graph shows the effect of the anti-aging essence composition on skin elasticity parameter (R2);

[0040] Figure 10 FIG1 is a test result graph showing the effect of the anti-aging essence composition on skin smoothness (SEsm);

[0041] Figure 11 The second test result graph shows the effect of the anti-aging essence composition on skin smoothness (SEsm);

[0042] Figure 12 The third test result graph shows the effect of the anti-aging essence composition on skin smoothness (SEsm);

[0043] Figure 13 This is a test result diagram of the effect of the anti-aging essence composition on skin wrinkle parameters (SEw);

[0044] Figure 14 The second test result graph shows the effect of the anti-aging essence composition on skin wrinkle parameters (SEw);

[0045] Figure 15 The third test result graph shows the effect of the anti-aging essence composition on skin wrinkle parameters (SEw);

[0046] Figure 16 This is a test result diagram of the effect of the anti-aging essence composition on skin wrinkle parameters (SEr);

[0047] Figure 17 The second test result graph shows the effect of the anti-aging essence composition on skin wrinkle parameters (SEr);

[0048] Figure 18 This is the third test result diagram of the effect of the anti-aging essence composition on skin wrinkle parameters (SEr). DETAILED DESCRIPTION

[0049] The present invention is further explained in detail below in conjunction with the drawings and descriptions of specific embodiments. However, the descriptions including the embodiments below are only intended to enable ordinary technicians in the technical field to which the present invention belongs to more clearly understand the principles and essence of the present invention, and are not intended to limit the present invention in any form.

[0050] Example 1

[0051] This embodiment provides an anti-aging freeze-dried powder composition, which is in the form of a freeze-dried powder and contains the following components measured by weight percentage on a dry basis:

[0052] C6-C3-C6 skeleton complex 10.0% to 75.0%;

[0053] β-Alanylhydroxyprolyldiaminobutyric acid benzylamine 10.0% to 65.0%;

[0054] Cationic amino acids 7.0% to 80.0%.

[0055] Furthermore, the above-mentioned C6-C3-C6 skeleton complex is one or more of quercetin, baicalin, myricetin, and luteolin.

[0056] The cationic amino acid is one or more of arginine, polyarginine, lysine and histidine.

[0057] With the continuous development of the cosmetics industry, all-natural skincare products are increasingly popular and sought after by consumers. Compared to skincare products made with chemically synthesized ingredients, skincare products containing natural extracts exhibit unique biological activities that are difficult to match with other ingredients. Furthermore, plant extracts are not only highly safe and gentle on the skin, but also demonstrate their unique skincare value. Leveraging these significant advantages, the present invention cleverly leverages the properties of natural plant extracts to successfully develop an anti-aging composition designed to provide consumers with a safer and more effective skincare experience.

[0058] The C6-C3-C6 skeleton complex used in this invention belongs to the class of natural flavonoid compounds, the most representative of which is quercetin, which is widely distributed in nature, such as in tea, coffee, apples, onions, and many other plants. Numerous studies have confirmed that quercetin not only possesses excellent antioxidant capacity but also effectively promotes cell apoptosis, showing great potential in the field of anti-aging. Numerous research results have revealed the significant advantages of using quercetin in combination with other drugs. For example, in 2018, researchers such as Rauf et al. demonstrated that the combination of quercetin and anticancer drugs can modulate the apoptotic pathways of cancer cells in vitro and in vivo, affecting their cell cycle. In 2019, a study by Sahyon et al. indicated that the combination of quercetin with other drugs can not only reduce side effects during treatment but also enhance the therapeutic effect. In 2013, a study by Azevedo et al. further demonstrated the application value of quercetin in the combination treatment of mental illness and other fields. In addition, the combination of rutin and quercetin has been shown to prevent painful peripheral neuropathy caused by oxaliplatin in mice. In 2021, researchers including Haoyang Zou found that the combination of quercetin and other drugs can significantly improve the treatment effect compared with using either drug alone.

[0059] As a lipophilic compound, quercetin can easily penetrate the cell membrane and activate multiple intracellular signal transduction pathways. It reduces the production of reactive oxygen species (ROS) through an electron exchange mechanism, thereby preventing ROS-mediated DNA damage and exerting anti-inflammatory effects by inhibiting the NF-κB signaling pathway. Researchers such as et al. have proposed that the combined use of quercetin and other anti-aging drugs can effectively eliminate senescent cells, providing a new idea and direction for the current anti-aging research using natural extracts.

[0060] Studies have shown that L-arginine is a source of nitric oxide (NO) in biological tissues, and its terminal guanidine group is produced by nitric oxide synthase (NOS), that is, L-arginine generates nitric oxide (NO) under the action of nitric oxide synthase (NOS), and NO is an important signaling molecule that plays a vital role in regulating the oxidative stress response in organisms. Oxidative stress is a cell damage phenomenon caused by excessive production of reactive oxygen species (ROS). Nitric oxide can react with ROS and neutralize its toxicity, thereby effectively reducing the damage caused to cells by oxidative stress. In addition, nitric oxide can further enhance the antioxidant defense capacity of cells by regulating the activity of antioxidant enzymes (such as superoxide dismutase). By generating nitric oxide, L-arginine helps regulate the redox state of cells and maintain the balance of oxidative and antioxidant systems in cells. Therefore, L-arginine has significant anti-stress properties, can effectively regulate the redox balance of cells, and has a positive effect on resisting oxidative stress.

[0061] The β-alanylhydroxyprolyldiaminobutyric acid benzylamine (hereinafter referred to as Erasin003) used in the present invention can effectively repair collagen damage caused by ultraviolet rays, stimulate the synthesis of type I collagen, promote the production of endogenous collagen, thereby significantly improving the state of photoaged cells and enhancing the activity of fibroblasts. Erasin003 and the above-mentioned L-arginine both contain a cation-π bond structure. This property enables it to significantly improve the solubility and stability of quercetin when combined with quercetin. In 2024, Lin Jiansheng and others successfully prepared arginine-quercetin-lipoic acid-Mn 2+ Self-assembling nanozymes, which work through multiple antioxidant mechanisms, have been applied to repair myocardial infarction damage. In the same year, Zhang Yinghua et al. proposed a method for preparing a composite emulsion for quercetin encapsulation. By optimizing the ratio of the components and determining the optimal formula, they successfully developed a composite emulsion system that significantly improved the antioxidant properties of quercetin after encapsulation.

[0062] In summary, the anti-aging freeze-dried powder composition provided in this embodiment uses three key active ingredients - a C6-C3-C6 skeleton complex, cationic amino acids, and Erasin003, to achieve an optimized synergistic effect and achieve excellent performance in antioxidant, anti-inflammatory, and anti-aging. Among them, the cationic amino acids and Erasin003 both contain cation-π bonds, which interact with quercetin through cation-π. This unique combination not only retains the original molecular structure of each active ingredient, but also significantly improves the bioavailability of quercetin. This combination not only enhances the efficacy of quercetin, but also gives the entire composition more powerful antioxidant, anti-inflammatory, and anti-aging capabilities, providing an unprecedented skin care experience for consumers who pursue healthy and youthful skin.

[0063] Example 2

[0064] This embodiment provides an anti-aging essence composition, which is in the form of an essence;

[0065] The composition comprises, by weight percentage of the total mass:

[0066] 0.05% to 0.75% of the anti-aging freeze-dried powder composition described in Example 1;

[0067] Solubilizer 5% to 10%;

[0068] Polyol 4% to 10%;

[0069] Oil 5% to 10%;

[0070] The rest is deionized water and preservatives.

[0071] Furthermore, the solubilizer is one or more of sorbitan monooleate polyoxyethylene ether (chemical name: polyoxyethylene sorbitan monooleate, alias: Tween 80, hereinafter referred to as Tween 80), glycerol polyether-26, Pluronic F127, and lecithin, and the mass fraction of the solubilizer is preferably 1% to 3%.

[0072] The polyol is one or more of butanediol, propylene glycol, pentanediol, hexanediol and ethoxydiglycol, and the mass fraction of the polyol is preferably 2% to 10%.

[0073] In some embodiments, the anti-aging serum composition further comprises carbon dioxide (CO2), which is added by continuously introducing CO2 into the composition until the composition reaches saturation. This CO2 saturation refers to continuously introducing CO2 into the anti-aging serum composition until the pH of the solution stabilizes within the range of 4.5 to 6.5 and maintains a certain equilibrium state.

[0074] Carbonated skincare is an emerging skincare concept and approach, centered around the effects of carbonic acid or carbon dioxide and its derivatives on the skin to achieve cosmetic benefits. Within the product system, the weak acidity of carbonic acid regulates the acid-base balance of skin cells, creating a more favorable environment for cell survival and metabolism. This enhances intracellular enzyme activity, improves cellular function, and helps maintain skin elasticity and resilience. Furthermore, carbon dioxide stimulates vasodilation in the skin, promoting blood circulation and enhancing the oxygen transport capacity of red blood cells, thereby participating in cellular metabolism and playing a positive role in delaying skin aging. In aqueous solution, CO2 can regulate skin pH and maintain skin homeostasis, thus possessing significant application value in skin care and the treatment of skin diseases. In skin care, Bock et al. found that treatment with a CO2 solution significantly reduced transepidermal water loss (TEWL) and pH. Fukagawa et al., through human studies and 3D epidermal model experiments, confirmed that transdermal CO2 solution application can improve flaking in dry skin and effectively alleviate scaling by mildly acidifying the stratum corneum. In the treatment of skin diseases, Schmidt J et al. have shown that high-concentration CO2 solutions can significantly promote wound and ulcer healing and inhibit the onset of Raynaud's syndrome. Furthermore, CO2-treated skin areas show an improvement rate of over 40% in scales and wrinkles. Long-term use of high-concentration CO2 solutions can significantly repair and regenerate the skin. These studies demonstrate the biological significance of aqueous CO2 solutions in skin repair and treatment.

[0075] Example 3

[0076] Based on the anti-aging freeze-dried powder composition provided in Example 1, the present invention also provides a preparation method thereof, comprising the following steps:

[0077] According to the above formula ratio, the C6-C3-C6 skeleton complex, cationic amino group and β-alanyl hydroxyprolyl diaminobutyric acid benzylamine are uniformly dissolved in deionized water according to weight fraction.

[0078] Furthermore, the C6-C3-C6 skeleton complex, cationic amino group and β-alanyl hydroxyprolyl diaminobutyric acid benzylamine are fully dissolved in deionized water by ultrasonication, and the ultrasonication operation temperature is 60-80°C.

[0079] Furthermore, the anti-aging composition can be placed in liquid nitrogen for rapid cooling and freezing, and finally freeze-dried to obtain an anti-aging freeze-dried powder composition.

[0080] Example 4

[0081] Based on the anti-aging essence composition provided in Example 2, the present invention also provides a preparation method thereof, comprising the following steps:

[0082] The anti-aging freeze-dried powder composition of Example 1 is fully dissolved in a solubilizing agent and a polyol by ultrasonication according to a weight ratio, and the operating temperature is 60-80° C. Then, oil is added, stirred uniformly, and then added to deionized water. After stirring and cooling, a preservative is added to obtain the anti-aging essence composition.

[0083] Furthermore, the preparation method further comprises injecting CO2 into the anti-aging essence composition to a saturated state, that is, introducing CO2 into the anti-aging essence composition until the pH of the solution is maintained at an equilibrium state of 4.5 to 6.5.

[0084] When the anti-aging composition is prepared as a freeze-dried powder, the freeze-dried powder needs to be converted into a solution system and then CO2 is introduced to a saturated state.

[0085] Example 5

[0086] Based on Examples 1-4, Examples 5 to 14 use experiments to illustrate the efficacy of the anti-aging composition provided by the present invention.

[0087] 0.08g of arginine, 0.02g of β-alanylhydroxyprolyldiaminobutyric acid benzylamine, and 0.20g of quercetin were dissolved in deionized water and sonicated at approximately 80°C to obtain a clear, transparent, pale yellow solution. The mixture was then rapidly frozen in liquid nitrogen for 30 minutes and freeze-dried to produce an anti-aging freeze-dried powder composition.

[0088] The lyophilized powder was dissolved in cell culture medium at a concentration of 100 μg / mL and HaCaT cells (also known as human immortalized keratinocytes) were incubated. It was found that compared with single quercetin and β-alanylhydroxyprolyldiaminobutyric acid benzylamine, the anti-aging composition had a cellular ROS clearance rate of 56.27%, which was much higher than the 28.21% and -8.4% of quercetin and β-alanylhydroxyprolyldiaminobutyric acid benzylamine, and had a more significant inhibitory ability on the expression of the cellular inflammatory factor IL-6, with an inhibition rate of 71.52%, showing a cellular anti-aging biological effect with combined regulatory intervention.

[0089] Example 6

[0090] 0.02g of arginine, 0.04g of β-alanylhydroxyprolyldiaminobutyric acid benzylamine, and 0.20g of quercetin were dissolved in deionized water and sonicated at approximately 80°C to obtain a clear, transparent, pale yellow solution. The mixture was then rapidly frozen in liquid nitrogen for 30 minutes and freeze-dried to produce a high-content, anti-aging, freeze-dried powder.

[0091] The lyophilized powder was dissolved in cell culture medium at a concentration of 100 μg / mL and the cells were incubated. It was found that compared with single quercetin and β-alanyl hydroxyprolyl diaminobutyric acid benzylamine, the anti-aging composition had a cellular ROS clearance rate of 26.49%, which was similar to or higher than the 28.21% and -8.4% of single quercetin and β-alanyl hydroxyprolyl diaminobutyric acid benzylamine. The effect on the expression of cellular inflammatory factors proved that it did not have a cellular anti-aging biological effect of combined regulatory intervention.

[0092] Example 7

[0093] 0.015g of arginine, 0.08g of β-alanylhydroxyprolyldiaminobutyric acid benzylamine, and 0.15g of baicalin were dissolved in deionized water and sonicated at approximately 80°C to obtain a clear, transparent, pale yellow solution. The mixture was then rapidly frozen in liquid nitrogen for 30 minutes and freeze-dried to produce a high-content, anti-aging, freeze-dried powder.

[0094] The lyophilized powder was dissolved in cell culture medium at a concentration of 100ug / mL and the cells were incubated. It was found that compared with single baicalin and β-alanylhydroxyprolyldiaminobutyric acid benzylamine, the anti-aging composition had a cell ROS clearance rate of 15.54%, which was lower than the 22.21% and -8.4% of single baicalin and β-alanylhydroxyprolyldiaminobutyric acid benzylamine. The effect on the expression of cellular inflammatory factors proved that it did not have the cellular anti-aging biological effect of combined regulatory intervention.

[0095] Example 8

[0096] 0.02g of arginine, 0.18g of β-alanylhydroxyprolyldiaminobutyric acid benzylamine, and 0.02g of baicalin were dissolved in deionized water and sonicated at approximately 80°C to obtain a clear, transparent, pale yellow solution. The mixture was then rapidly frozen in liquid nitrogen for 30 minutes and freeze-dried to produce a high-content, anti-aging, freeze-dried powder.

[0097] The lyophilized powder was dissolved in cell culture medium at a concentration of 100ug / mL and the cells were incubated. It was found that compared with single baicalin and β-alanylhydroxyprolyldiaminobutyric acid benzylamine, the anti-aging composition had a cell ROS clearance rate of -8.4%, which was 22.21% and -8.4% lower than single baicalin and β-alanylhydroxyprolyldiaminobutyric acid benzylamine, respectively. The effect on the expression of cellular inflammatory factors proved that it did not have a cellular anti-aging biological effect of combined regulatory intervention.

[0098] Example 9

[0099] 0.02g of arginine, 0.20g of β-alanylhydroxyprolyldiaminobutyric acid benzylamine, and 0.02g of baicalin were dissolved in deionized water and sonicated at approximately 80°C to obtain a clear, transparent, pale yellow solution. The mixture was then rapidly frozen in liquid nitrogen for 30 minutes and freeze-dried to produce a high-content, anti-aging, freeze-dried powder.

[0100] The lyophilized powder was dissolved in cell culture medium at a concentration of 100 μg / mL and the cells were incubated. It was found that compared with single baicalin and β-alanylhydroxyprolyldiaminobutyric acid benzylamine, the lyophilized powder combination had a cell ROS clearance rate of -12.94%, which was 28.21% and -8.4% lower than single baicalin and β-alanylhydroxyprolyldiaminobutyric acid benzylamine, respectively. The effect on the expression of cellular inflammatory factors proved that it has a cellular anti-aging biological effect of combined regulatory intervention.

[0101] Table 1 Composition and content of each component of the anti-aging composition provided in Examples 5-9

[0102]

[0103] Example 10

[0104] Based on the five anti-aging compositions provided in Examples 5 to 9, in this example, the cytotoxicity (CCK-8) test of the active ingredient combination system was performed on the five anti-aging compositions.

[0105] The experimental method is as follows:

[0106] (1) Plating: Add trypsin to the HSF culture flask for 2 min, add 2% culture medium to terminate the digestion, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend with 10% culture medium, take 90 μL of cell fluid and 10 μL of trypan blue, add the counting plate technology, calculate the dilution multiple, plate, and culture in an incubator for 2 days.

[0107] (2) Sample Addition: Dissolve the anti-aging lyophilized powder composition of Examples 5-9 in 5% culture medium to a concentration of 1 mg / mL. Discard the liquid in the well plate and add the sample. Set up a negative control group and culture in an incubator for 24 hours.

[0108] (3) Adding the detection agent: dilute the CCK-8 detection agent to 10% with 5% culture medium, then discard the liquid in the well plate, wash the well plate with PBS, aspirate the PBS and add the diluted CCK-8 detection agent, set up a blank control, and then incubate for 0.5 h.

[0109] (4) Detection: Detect the absorbance at 450 nm using an enzyme-labeled instrument.

[0110] The CCK-8 experiment in this example detected the cytotoxicity of the combined active ingredients to HaCaT cells, and the cytotoxicity was determined based on the cell survival rate. Figure 1 It can be seen that the cell survival rates of Examples 5-9 are all above 90%, and there is no significant difference compared with the blank control, so Examples 5-9 are non-cytotoxic.

[0111] Example 11

[0112] Based on the five anti-aging compositions provided in Examples 5 to 9, in this example, the five anti-aging compositions were subjected to an intracellular antioxidant effect (CAA) test of the active ingredient combination system.

[0113] The experimental method is as follows:

[0114] (1) Plating: 100 μL of HaCaT cells were seeded into a black 96-well plate at a concentration of 1.5×10 5 cells / mL and cultured continuously in an incubator (37° C., 5% CO 2 ).

[0115] (2) The culture medium was removed and the plate was washed twice with 200 μL of serum-free (FBS-free) culture medium. Then, 50 μL of 200 μmol / L DCFH-DA was added to each well.

[0116] (3) Samples were prepared according to Examples 5-9, and 50 μL of samples of different concentrations were added to each of the blank and positive control groups without samples. 50 μL of serum-free culture medium was added to the blank and positive control groups, and the cells were incubated at 37°C for 1 h.

[0117] (4) Carefully discard the culture medium and wash twice with 200 μL PBS. Then, add 100 μL PBS to the no-sample group and 100 μL 6 mmol / L AAPH to the positive control group and the sample group.

[0118] (5) Use a microplate reader to measure the fluorescence intensity at 480 nm excitation / 530 nm emission, measure the fluorescence intensity every 60 s, and detect for 1 h.

[0119] (6) Calculation of intracellular antioxidant capacity:

[0120] CAA Units=100-(AUCsample-AUCsample background) / (AUCctrl-AUCctrlbackground)

[0121] The CAA experiment in this example detected the effect of the combined active ingredients on the production of ROS in HaCaT cells. ROS are produced by peroxyl radicals induced by AAPH, which cause the cells to be oxidized by non-luminescent DCFH into fluorescent 2',7'-dichlorofluorescein (DCF), thereby showing the antioxidant activity of the cells. Figure 2 It can be seen that the triple synergistic active combination of quercetin: arginine: Erasin003 in Example 5 achieves the best antioxidant effect when the ratio is 9:1:4 wt%.

[0122] Example 12

[0123] Based on the five anti-aging compositions provided in Examples 5 to 9, in this example, CO2 was added to the anti-aging composition system of Example 5 to measure the inflammatory factors in HaCaT cells.

[0124] The experimental method is as follows:

[0125] (1) Plating: Add trypsin to the HaCaT culture flask and digest for 4 minutes. Add 2% culture medium to terminate the digestion. Centrifuge at 1000 rpm for 3 minutes and discard the supernatant. Resuspend with 10% culture medium, take 90 μL of cell fluid + 10 μL of trypan blue and add it to the counting plate. Calculate the dilution multiple and plate it. Place in the incubator and culture for 2 days.

[0126] (2) Sample addition and CO2 flow: At room temperature and pressure, CO2 was introduced into the culture medium at a flow rate of 5 smmL using a gas flowmeter until saturation (pH 6.5) was achieved to obtain a CO2-synergistic three-combination system, named the three-synergistic composition@CO2. Arginine, Erasin003, quercetin, the three-synergistic composition (i.e., the anti-aging composition without CO2 injection), and the three-synergistic composition@CO2 were added to a 96-well plate. A positive control and 100 ng / mL dexamethasone were set as controls and stimulated for 2 h.

[0127] (3) Add TNF-α: dilute the pro-inflammatory factors TNF-α:IFN-γ in a 5% culture medium at a ratio of 1:1 to 50 ng / mL, add 10 μL of the pro-inflammatory factor mixture to each well, and culture in an incubator for 24 h.

[0128] (4) Enzyme-Linked Immunosorbent Assay: Cell supernatants were collected and assayed using an ELISA kit according to the manufacturer's instructions. The immunological response was qualitatively and quantitatively determined by the specificity of antigen-antibody binding to detect the amount of IL-6 secreted by cells stimulated by the active composition screened by the in vitro biochemical method, thereby determining the anti-inflammatory effect of the active composition.

[0129] The IL-6 expression level is used as an evaluation index for anti-inflammatory ability. The lower the IL-6 expression level, the stronger the anti-inflammatory ability. Figure 3It can be seen that when the triple synergistic composition system of quercetin: arginine: Erasin003 ≈ 9:1:4wt% was compounded and CO2 was introduced (denoted as triple synergistic composition@CO2), the expression level of IL-6 in the triple synergistic composition@CO2 was significantly reduced compared with the positive control, and compared with single arginine, Erasin003 and quercetin, the expression level of IL-6 in the triple synergistic composition@CO2 showed a lower expression, which had a better anti-inflammatory effect.

[0130] Example 13

[0131] Based on Example 5 of the five anti-aging compositions provided in Examples 5 to 9, the expression of inflammation-related genes (ICAM, VCAM, MMP-1 mRNA) in HSF cells using the CO2-filled anti-aging composition, i.e., the triple synergistic composition @CO2, was measured (RT-qPCR).

[0132] The experimental method is as follows:

[0133] (1) Total RNA extraction

[0134] (a) For the single component and the three synergistic combination active ingredients and the treated HSF cells, add 300 μL of lysis buffer and gently pipette 5-10 times until the suspended solids are dissolved and the solution is clear. Transfer to a clean centrifuge tube.

[0135] (b) Add an equal volume of binding buffer to the lysate and gently invert to mix.

[0136] (c) Transfer the mixture to a purification column, centrifuge at 12,000 × g for 30 seconds, and discard the liquid in the collection tube.

[0137] (d) Add 300 μL of washing solution 1, centrifuge at 12,000 × g for 30 seconds, and discard the liquid in the collection tube.

[0138] (e) Add 300 μL of washing solution 2, centrifuge at 12,000 × g for 30 seconds, and discard the liquid in the collection tube.

[0139] (f) Repeat step (e) once.

[0140] (g) Centrifuge at maximum speed for 2 minutes and remove the liquid from the tube.

[0141] (h) Place the purification column in an elution tube, add 40 μL of elution buffer, incubate at room temperature for 2 minutes, and centrifuge at maximum speed for 30 seconds. The resulting solution is the purified RNA.

[0142] (2) RNA concentration determination: The collected RNA solution was used as a reference with sterile, enzyme-free ddH2O to measure the absorbance at 260 nm and 280 nm of each group. If the OD260 / OD280 ratio was between 1.8 and 2.0, it could be used for the next step.

[0143] (3) Synthesis of first-strand cDNA: Add the purified RNA to a centrifuge tube according to the dosage indicated in the Beyotime kit, mix gently, and centrifuge. Place the centrifuge tube in a PCR instrument and set the program to incubate at 25°C for 10 minutes, incubate at 42°C for 60 minutes, and heat at 85°C for 10 minutes. Store the resulting cDNA at -20°C.

[0144] (4) RT-PCR reaction system and conditions

[0145] ① Prepare upper and lower primers: ICAM, VCAM, MMP-1

[0146] ②Reaction system: BeyoFast SYBR Green qPCR Mix (2X) 10μL+Forward and ReversePrimer Mix 2μL+Template DNA 2μL+RNase-Free Water 6μL

[0147] ③Reaction conditions:

[0148] a. Pre-denaturation: 95℃ for 2 minutes

[0149] b. Denaturation: 95℃ for 15 seconds

[0150] c. Annealing / extension: 50-60°C for 15 seconds.

[0151] Repeat steps b and c: 40 cycles total

[0152] (5) Analyze the results using Lightcycle 480 software.

[0153] RT-qPCR is used to measure the effect of the triple synergistic composition @CO2 on the expression of aging-related genes in HSF cells, thereby exploring the molecular mechanism of the anti-aging effect of the composition on HSF cells and screening the optimal triple synergistic combination ratio. Figure 4-6 It can be seen that when the freeze-dried powder composition of quercetin: arginine: Erasin003 ≈ 9:1:4wt% was compounded and CO2 was introduced, ICAM, VCAM, and MMP-1 were significantly reduced, and all had a down-regulating performance, indicating that the triple synergistic composition @CO2 has a better anti-aging effect, that is, the lower the expression level of various factors, the better the effect.

[0154] Example 14

[0155] Based on the five anti-aging compositions provided in Examples 5 to 9, in this example, the human efficacy evaluation of the freeze-dried powder composition@CO2 essence system was performed.

[0156] The freeze-dried powder composition made of the anti-aging composition is added to the essence. The essence formula is shown in Table 2, and a carbon dioxide aerosol manufacturing system is used to add carbon dioxide to cosmetics. For example, the method of the previous patent CN201810645213.8 of the inventor is used, and the relevant content of CN201810645213.8 is introduced herein in full. Chinese healthy subjects aged between 30 and 45 years old are selected and carried out under the method of control (self-control before and after using the sample). Before the subject used the test product 0D, the skin elasticity, wrinkle parameters, skin roughness and other parameters of the corners of the mouth and eyes of the product 14D and 28D were measured, and the rate of change of each parameter within 28 days was calculated by the formula.

[0157] Change rate % = (D28-D0)D0*100%

[0158] Table 2 Test sample information

[0159]

[0160] (1) Test Example 1—Skin Elasticity Test Results

[0161] The statistical analysis results of the smoothness test data before and after the product is used at 0D, 14D, and 28D are shown in the table below. Figure 7-9 Compared with 0D before using the product, after the subjects used the product for 28D, the skin elasticity of the corner of the eye was increased relative to the initial R2. The control, the three-synergistic composition (abbreviated as: three-synergistic, the same below), and the three-synergistic composition @CO2 (abbreviated as: three-synergistic@CO2, the same below) all showed the performance of increasing skin elasticity. Among them, the average R2 change rate of the three groups increased by 28.39%, 30.44% and 31.62%, respectively, showing a gradually increasing trend. Compared with the initial R2, the skin of the corner of the eye showed a decrease in elasticity in the control, and the average R2 change rate of the three-synergistic and three-synergistic@CO2 increased by -9.35%, 6.97% and 15.55%, respectively, showing a gradually increasing trend.

[0162] (2) Test Example 2—Skin Smoothness Test Results

[0163] The statistical analysis results of the smoothness test data before and after the product is used at 0D, 14D, and 28D are shown in the table below. Figure 10-12Compared with the initial SEsm before product use (0 days), after 28 days of product use, the SEsm values ​​of the skin at the corners of the eyes decreased in the control, three-synergistic, and three-synergistic@CO2 groups relative to the initial SEsm. The average SEsm change rates of the three groups increased by 1.67%, 15.37%, and 19.04%, respectively, showing a trend of gradually decreasing skin smoothness, but no significant difference was achieved. The SEsm values ​​of the skin at the corners of the mouth decreased in the control, three-synergistic, and three-synergistic@CO2 groups relative to the initial SEsm. The average SEsm change rates of the three groups decreased by 24.39%, 15.77%, and 14.15%, respectively. The experimental group did not show a trend of gradually decreasing skin smoothness.

[0164] (3) Test Example 3—Skin Wrinkle Parameter Test Results

[0165] The statistical analysis results of the smoothness test data before and after the product is used at 0D, 14D, and 28D are shown in the table below. Figure 13-15 Compared with the 0 day before using the product, after using the product for 28 days, the Sew of the eye corner skin in the control, triple synergistic, and triple synergistic@CO2 groups decreased relative to the initial eye corner skin. The average Sew change rates of the three groups decreased by 20.66%, 26.13%, and 27.22%, respectively, showing a trend of gradually reducing skin wrinkles, but no significant difference was reached; the SEw of the mouth corner wrinkles in the control, triple synergistic, and triple synergistic@CO2 groups decreased relative to the initial skin. The average Sew change rates of the three groups decreased by 28.98%, 31.59%, and 34.39%, respectively, showing a trend of gradually reducing skin wrinkles.

[0166] (4) Test Example 4—Skin Roughness Parameter Test Results

[0167] The statistical analysis results of the smoothness test data before and after the product is used at 0D, 14D, and 28D are shown in the table below. Figure 16-18 Compared with the 0 day before using the product, after using the product for 28 days, the SEr of the eye corner skin of the subjects was reduced compared with the initial eye corner skin, and compared with the initial skin, the SEr of the control, triple synergistic, and triple synergistic@CO2 groups were all reduced, among which the average SEr change rates of the three groups decreased by 23.85%, 22.50%, and 22.40%, respectively, which did not reach significant differences; SEr of mouth corner wrinkles: Compared with the initial skin, SEr of the control, triple synergistic, and triple synergistic@CO2 groups were all reduced, among which the average SEr change rates of the three groups decreased by 21.45%, 24.06%, and 24.82%, respectively.

[0168] In summary, in the four tests of human efficacy evaluation, anti-aging indicators (skin elasticity, smoothness, and wrinkle parameters) showed that compared with original skin, the average values ​​of the Triple Synergy and Triple Synergy@CO2 Essence groups showed a trend of gradual and potential improvement in aging, among which Triple Synergy@CO2 showed a slight advantage.

[0169] It should be noted that all the above measurements were performed on at least three freshly prepared samples, and their average values ​​and standard deviations were reported.

[0170] Example 15

[0171] Based on the above Examples 5 to 14, the anti-aging composition provided by the present invention can be applied to skin care products and added to the skin care products in an appropriate amount.

[0172] This anti-aging composition is specifically designed for anti-aging needs and has undergone rigorous testing to ensure its safety and effectiveness. It can be incorporated into various skincare formulas, significantly enhancing their anti-aging benefits by simply adding the recommended dosage.

[0173] In addition, the use of skin care products containing the anti-aging composition has the potential to enhance the elasticity and firmness of the customer's skin, smooth the skin, reduce wrinkles, and improve the roughness of the skin.

[0174] Example 16

[0175] The present invention also provides a skin care product, comprising the anti-aging freeze-dried powder composition provided by Example 1, or the anti-aging essence composition provided by Example 2, or the anti-aging freeze-dried powder composition prepared by the preparation method provided by Example 3, or the anti-aging essence composition prepared by the preparation method provided by Example 4.

[0176] The ideal embodiment of the present invention is inspiration. Through the above description, relevant personnel can make various changes and modifications without departing from the scope of the technical idea of ​​this invention.

[0177] The technical scope of this invention is not limited to the contents of the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. An anti-aging freeze-dried powder composition, characterized in that: The composition is in the form of a freeze-dried powder and contains the following components measured in percentage by weight on a dry basis: C6-C3-C6 skeleton complex 10.0% to 75.0%; β-Alanylhydroxyprolyldiaminobutyric acid benzylamine 10.0% to 65.0%; Cationic amino acids 7.0% to 80.0%.

2. The anti-aging freeze-dried powder composition according to claim 1, characterized in that The C6-C3-C6 skeleton complex is one or more of quercetin, baicalin, myricetin, and luteolin; and / or, The cationic amino acid is one or more of arginine, polyarginine, lysine and histidine.

3. An anti-aging essence composition, characterized in that: The composition is in the form of an essence liquid; The composition comprises, by weight percentage of the total mass: The rest is deionized water and preservatives.

4. The anti-aging essence composition according to claim 3, characterized in that The solubilizing agent is one or more of sorbitan monooleate polyoxyethylene ether, glycerol polyether-26, Pluronic F127, and lecithin; and / or, The polyol is one or more of butanediol, propylene glycol, pentanediol, hexanediol and ethoxydiglycol.

5. The anti-aging essence composition according to claim 3, characterized in that Also includes: CO2, The CO2 is in a saturated state in the anti-aging essence composition.

6. A method for preparing an anti-aging freeze-dried powder composition, characterized in that the steps include: The C6-C3-C6 skeleton complex, cationic amino acids and β-alanylhydroxyprolyldiaminobutyric acid benzylamine are fully dissolved in deionized water by ultrasonic method according to the weight ratio, and the operating temperature is 60-80°C. The mixed solution is then placed in liquid ammonia and rapidly cooled and frozen. Finally, the anti-aging freeze-dried powder composition is obtained by freeze-drying.

7. A method for preparing an anti-aging essence composition, characterized in that the steps include: The anti-aging freeze-dried powder composition according to claim 1 or 2 is fully dissolved in a solubilizing agent and a polyol by ultrasonic method according to a weight ratio, and the operating temperature is 60-80°C. Then, oil is added, stirred uniformly, added to deionized water, stirred to cool, and a preservative is added to obtain the anti-aging essence composition.

8. The preparation method according to claim 7, characterized in that The anti-aging essence composition is charged with CO2 until saturated.

9. Application of an anti-aging composition in skin care products, characterized in that: The anti-aging composition is the anti-aging freeze-dried powder composition according to claim 1 or 2 or the anti-aging essence composition according to any one of claims 3 to 5.

10. A skin care product, characterized in that: The invention comprises the anti-aging freeze-dried powder composition according to claim 1 or 2, or the anti-aging essence composition according to any one of claims 3 to 5, or the anti-aging freeze-dried powder composition prepared by the preparation method according to claim 6, or the anti-aging essence composition prepared by the preparation method according to claim 7 or 8.

Images