Application of isoflavone compound in freckle removal and whitening, anti-inflammation, anti-oxidation, anti-wrinkle and anti-aging of skin

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of an isoflavone compound in freckle removal and whitening, anti-inflammation, anti-oxidation, anti-wrinkle and anti-aging of skin. The invention provides a composition for skin, the effective component of the composition is calycosin-7-glucoside, and the composition not only has the total antioxidant capacity of remarkably scavenging free radicals, but also remarkably inhibits the expression and activity of tyrosinase and formation of melanin, has the application potential of whitening skin and fading spots, provides a new raw material selection for whitening products, and has wide application prospects. Meanwhile, the composition also has excellent anti-inflammatory, anti-oxidation and anti-aging effects, and an excellent theoretical basis is provided for the preparation of functional cosmetics.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine technology, and more specifically relates to the application of a potential isoflavone compound in skin freckle removal, whitening, anti-inflammatory, anti-oxidation, anti-wrinkle and anti-aging. Background Art

[0002] Spot removal, whitening, anti-inflammatory and anti-oxidation, anti-wrinkle and anti-aging have always been the core needs of skin care. With the promotion of the concept of natural skin care, cosmetics containing natural plant-derived ingredients are also more popular among consumers. Plant active ingredients play an important role in skin spot removal, whitening, anti-inflammatory and anti-oxidation, anti-wrinkle and anti-aging because of their wide sources, high safety, and the possibility of multi-target effects.

[0003] Research has found that spots and blemishes are one of the main reasons for dull skin. Whitening can reduce spots and blemishes, making the skin more even and smooth. The mechanism of action of whitening products is mainly to inhibit the synthesis of melanin and promote the metabolism of melanin, thereby reducing the accumulation of melanin and achieving the whitening effect.

[0004] In addition to genetic factors, skin aging is affected by many external factors. For example, ultraviolet rays can cause cells to produce excessive free radicals, which exceed the cells' antioxidant capacity and cause oxidative stress, thereby accelerating skin aging. It can be seen that inhibiting the generation of free radicals and removing excessive free radicals can prevent cell oxidative damage and alleviate skin aging.

[0005] As people's demand for functional cosmetics grows, safer new raw materials are also sought after. Therefore, finding a safer and more effective plant extract provides a new option for skin care products and has become an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] The purpose of the present invention is to provide an application of potential isoflavone compounds in skin freckle removal and whitening, anti-inflammatory and antioxidant, anti-wrinkle and anti-aging effects, so as to solve the problems existing in the above-mentioned prior art.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] One of the technical solutions of the present invention is to provide a skin composition, wherein the active ingredient of the skin composition includes calycosin isoflavone glycosides.

[0009] Furthermore, the skin composition also includes a solvent.

[0010] Optionally, the solvent includes one or more of phosphate buffered saline (PBS), dimethyl sulfoxide (DMSO), sterile water, physiological saline, propylene glycol, polyethylene glycol, glycerol or vegetable oil.

[0011] Furthermore, the concentration of the calycosin in the skin composition is 0.1 to 20 wt %.

[0012] It should be noted that the concentration of calycosin in the cell experiment in the specific embodiment of the present invention is low, but it can be used in cosmetics at a higher concentration because:

[0013] Cell experiments are usually conducted to explore the mechanism of action of a specific ingredient, evaluate its biological activity, and determine the safe and effective concentration range. In this process, excessively high concentrations may cause cytotoxicity and affect the interpretation of experimental results. Therefore, lower concentrations are used in cell experiments to observe and analyze the effects of ingredients.

[0014] Human skin has a natural barrier - the stratum corneum, which can effectively prevent most substances from directly penetrating into the body. This means that even if a higher concentration of calycosin is used in cosmetics, the amount that can actually penetrate deep into the skin and exert an effect may be much lower than the actual amount added. This is also why calycosin can appear in higher concentrations in cosmetics without causing serious side effects or toxicity.

[0015] Therefore, the difference in concentration between cell experiments and cosmetic applications is mainly due to the differences in the physiological characteristics of cells and skin tissues. It cannot be assumed that the concentration that is toxic in cell experiments is not suitable for cosmetics.

[0016] The second technical solution of the present invention is to provide an application of the above-mentioned skin composition in daily chemical products.

[0017] Optionally, the application includes one of lotion, skin softener, toner, astringent, nourishing cream, eye cream, liquid foundation, dry powder, essence, oil, gel, lotion, beauty serum, facial mask, facial mask base liquid, soap, cleansing cream, cleansing foam and body cleanser.

[0018] The third technical solution of the present invention is to provide a use of calycosin as an effective ingredient in whitening and skin care cosmetics.

[0019] The fourth technical solution of the present invention is to provide an application of calycosin in cosmetics for inhibiting cellular melanin production.

[0020] The fifth technical solution of the present invention is to provide a use of calycosin in inhibiting the expression of tyrosinase and in active cosmetics.

[0021] Technical solution six of the present invention: Provides an application of calycosin in anti-inflammatory and antioxidant cosmetics.

[0022] Technical solution seven of the present invention: provides an application of calycosin isoflavone glycosides in anti-aging cosmetics.

[0023] The present invention discloses the following technical effects:

[0024] The composition comprising calycosin glycosides of the present invention has a significant total antioxidant capacity of scavenging free radicals.

[0025] The composition containing calycosin glycosides of the present invention has the ability to significantly inhibit the formation of melanin and has obvious tyrosinase inhibition ability.

[0026] The composition containing calycosin isoflavone glycosides of the present invention has application potential for skin whitening and spot removal, and provides a new raw material selection for whitening products.

[0027] The composition containing calycosin glycosides of the present invention has anti-inflammatory application potential.

[0028] The composition containing calycosin glycosides of the present invention has potential application in anti-wrinkle and anti-aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0030] Figure 1 This is the antioxidant activity diagram of calycosin isoflavone glycosides and α-arbutin detected by ABTS method;

[0031] Figure 2 This is the antioxidant activity diagram of calycosin and α-arbutin detected by FRAP method;

[0032] Figure 3 is the inhibition rate of α-arbutin and calycosin on mushroom tyrosinase, where a is α-arbutin and b is calycosin;

[0033] Figure 4 The results show the effect of DMSO on the viability of B16F10 cells;

[0034] Figure 5 The results are the effects of α-arbutin and calycosin on the proliferation and vitality of B16F10 cells, wherein (a) is α-arbutin, and (b) is calycosin;

[0035] Figure 6 The inhibition of tyrosinase expression in B16F10 cells by calycosin isoflavone glycosides;

[0036] Figure 7 This is the inhibitory effect of calycosin on the expression of β-galactosidase in photoaged cells. DETAILED DESCRIPTION

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] Astragalus is the root of Astragalus membranaceus and Astragalus membranaceus of the Leguminosae family. Its root has a long history of being used as medicine. Many ancient medical books believe that it has the effect of invigorating qi and replenishing deficiency.

[0043] Inner Mongolia Astragalus, also known as Zhengbeiqi, is a plant with significant medicinal value. It is cold in nature and slightly warm in nature, and enters the lung and spleen meridians.

[0044] The main effects include: tonifying Qi. Astragalus is known as the best Qi tonifier among all medicines. It is suitable for people with spleen and stomach Qi deficiency who usually have Qi deficiency, fatigue, poor appetite and loose stools. It is also suitable for people with weak constitution and frequent colds. It is used to improve the body's immunity and eliminate fatigue; nourishing blood. It tonifies Qi and promotes blood circulation at the same time. It is suitable for people with blood deficiency and sallow complexion, as well as people with symptoms such as arthralgia caused by Qi deficiency and inability of blood to nourish the tendons and veins, sequelae of stroke, numbness of the skin, hemiplegia, etc.; consolidating the exterior and stopping sweating. It is suitable for people of all ages with exterior deficiency and spontaneous sweating due to Qi deficiency; promoting diuresis and reducing swelling. It can be used to treat urinary problems and has the effect of eliminating edema; detoxifying, draining pus, astringing sores and promoting tissue regeneration. It is suitable for patients in the middle stage of sores, poisoning, deficiency of the body and excessive toxicity, inability to ulcerate, and physical weakness and long-term sores that cannot be astringed.

[0045] Processing and usage: When used raw, it has the effects of invigorating Qi and strengthening the exterior, promoting diuresis and reducing swelling, detoxifying and regenerating tissue. It is suitable for symptoms such as spontaneous sweating, night sweats, blood stasis, edema or ulcers that do not heal for a long time; roasted with honey, it has the effects of invigorating Qi, nourishing blood and benefiting the middle, and is suitable for patients with internal injuries, fatigue, spleen deficiency and diarrhea, Qi deficiency, blood deficiency and Qi deficiency.

[0046] How to eat: Soak in water and drink, to absorb its medicinal effects quickly and conveniently; stew it in soup to better exert its nourishing effect.

[0047] Calycosin-7-O-β-D-glucoside, also known as calycosin, is an isoflavone isolated from Astragalus. Calycosin-7-O-β-D-glucoside has multiple biological activities, such as neuroprotection, cardioprotection and anti-inflammatory effects.

[0048] The main biological activities and pharmacological effects of calycosin isoflavone glycosides:

[0049] Cardioprotective effect: Calycosin isoflavone glycosides have excellent performance in cardioprotection. They can protect myocardial cells from ischemia-reperfusion injury, help maintain heart function, and reduce the risk of heart attack.

[0050] Neuroprotective effect: The compound also shows a certain neuroprotective effect, which can reduce the death of nerve cells and promote nerve regeneration, and may be beneficial for the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease;

[0051] Antiviral effect: Calycosin has an inhibitory effect on certain viruses (such as Coxsackie virus) and can be used as an adjuvant treatment for viral diseases.

[0052] Immunomodulatory effect: As a natural phytoestrogen, calycosin can bind to estrogen receptors in the human body to produce estrogen-like effects. It has obvious immunomodulatory function and helps to enhance the body's immunity.

[0053] Application fields of calycosin isoflavone glycosides:

[0054] Pharmaceutical industry: Due to its multifaceted pharmacological activities, calycosin isoflavone glycosides are widely used in new drug research and development, especially in the fields of cardiovascular diseases, neurodegenerative diseases, and immune system diseases.

[0055] Health Food: As a natural ingredient, it is also added to various health foods to enhance the nutritional value and health benefits of the products.

[0056] In order to ensure the effectiveness and stability of calycosin isoflavone glycosides, it is generally recommended to store them under low temperature conditions (2-8°C) and avoid prolonged exposure to air.

[0057] To improve the stability of whitening compositions:

[0058] In some optional embodiments, the skin composition includes a moisturizer in addition to the calycosin and the solvent. The optional moisturizer includes at least one of hyaluronic acid, butylene glycol and trehalose.

[0059] Among them, the amount of hyaluronic acid used is 0.05% to 0.5% of the sum of the mass of the calycosin isoflavone glycosides and the solvent, and the commonly used amount is 1%; the amount of butanediol used is 3% to 10% of the sum of the mass of the calycosin isoflavone glycosides and the solvent, and the commonly used amount is 5%; the amount of trehalose used is 1% to 5% of the sum of the mass of the calycosin isoflavone glycosides and the solvent, and the commonly used amount is 2%.

[0060] Hyaluronic acid is a very mild moisturizer with strong moisturizing ability. It can maintain the moisture balance of the skin and is almost non-irritating. Butylene glycol has a good moisturizing effect and can improve the skin feel of the product. It is gentle on the skin and has minimal irritation. Trehalose has good moisturizing and stabilizing effects, which can protect the active ingredients from environmental influences. It is gentle on the skin and has minimal irritation.

[0061] In some optional embodiments, the skin composition includes a stabilizer in addition to the calycosin glycosides and the solvent. The optional stabilizer includes at least one of polyvinyl alcohol, carbomer and xanthan gum.

[0062] Among them, the amount of polyvinyl alcohol used is 0.5% to 2% of the sum of the mass of the isoflavone glycosides and the solvent, and the commonly used amount is 1%; the amount of carbomer used is 0.5% to 1.5% of the sum of the mass of the isoflavone glycosides and the solvent, and the commonly used amount is 0.5% to 1%; the amount of xanthan gum used is 0.1% to 1% of the sum of the mass of the isoflavone glycosides and the solvent, and the commonly used amount is 0.5%.

[0063] Polyvinyl alcohol can form a protective film to prevent active ingredients from direct contact with the external environment. It is gentle on the skin and has minimal irritation. Carbomer can increase the viscosity of the product, improve skin feel, and is gentle on the skin and has minimal irritation. Xanthan gum has good thickening and suspending effects, which can improve the stability of the product. It is gentle on the skin and has minimal irritation.

[0064] In some optional embodiments, the skin composition includes a chelating agent in addition to the calycosin glycosides and the solvent. The optional chelating agent includes ethylenediaminetetraacetic acid (EDTA) or ethylenediamine-N,N'-disuccinic acid (EDDS).

[0065] The dosage of EDTA is 0.05% to 0.2% of the total mass of the calycosin isoflavone glycosides and the solvent, and the commonly used dosage is 0.1%; the dosage of EDDS is 0.05% to 0.2% of the total mass of the calycosin isoflavone glycosides and the solvent, and the commonly used dosage is 0.1%.

[0066] EDTA is a commonly used chelating agent that can form stable complexes with a variety of metal ions to reduce the catalytic effect of metal ions. Although EDTA itself is less irritating to the skin, it may cause slight irritation at high concentrations. Therefore, attention should be paid to concentration control when using it, and it must be used within the specified range of the present invention. EDDS is a more environmentally friendly chelating agent that also has good chelating effect and is extremely less irritating to the skin.

[0067] In some optional embodiments, the skin composition includes a UV absorber in addition to the calycosin and the solvent. The optional UV absorber includes titanium dioxide or cyclohexasiloxane.

[0068] Among them, the amount of titanium dioxide used is 2% to 5% of the total mass of the calycosin isoflavone glycosides and the solvent, and the commonly used amount is 2% to 3%; the amount of cinquefoxione used is 2% to 5% of the total mass of the calycosin isoflavone glycosides and the solvent, and the commonly used amount is 2% to 3%.

[0069] Titanium dioxide is a physical sunscreen that can reflect ultraviolet rays, is mild to the skin and has minimal irritation. It should be noted that the safety of nano-titanium dioxide should be ensured when used; cyclohexasiloxane is a chemical sunscreen that can absorb ultraviolet rays, is mild to the skin and has minimal irritation.

[0070] In some optional embodiments, the skin composition includes a preservative in addition to the calycosin isoflavone glycosides and a solvent. Optional ultraviolet absorbers include phenoxyethanol, parabens (methylparaben, propylparaben, etc.) or potassium sorbate.

[0071] Among them, the amount of phenoxyethanol used is 0.1% to 0.4% of the sum of the mass of the calycosin isoflavone glycosides and the solvent, and the commonly used amount is 0.2% to 0.3%; the amount of parabens used is 0.1% to 0.4% of the sum of the mass of the calycosin isoflavone glycosides and the solvent, and the commonly used amount is 0.2% to 0.3%; the amount of potassium sorbate used is 0.1% to 0.5% of the sum of the mass of the calycosin isoflavone glycosides and the solvent, and the commonly used amount is 0.2%.

[0072] The above scheme can effectively improve the storage stability of the composition containing calycosin at room temperature, while ensuring its mildness and low irritation to the skin and complying with national regulations.

[0073] The synthesis pathway of melanin mainly goes through two stages: first, L-tyrosine in melanocytes is hydroxylated to DOPA as a substrate under catalysis, and DOPA continues to be a reaction substrate of tyrosinase to further generate dopaquinone, which reacts through two different routes to generate eumelanin and pheomelanin. When glutathione and cysteine ​​are present in the cellular environment, dopaquinone will react with them to generate glutathione DOPA and cysteinyl DOPA. Glutathione DOPA can be further converted into pheomelanin through multiple steps. When glutathione is absent or present in small amounts, When glutathione and cysteine ​​are reacted, dopaquinone generated by tyrosinase is further oxidized to dopachrome, and then self-cyclized and decarboxylated to form 5,6-dihydroxyindole (DHI), which generates 5,6-indolequinone (IQ) under the action of tyrosinase, or is converted into 5,6-dihydroxyindole-2-carboxylic acid (DHICA) under the action of tyrosinase-related protein 2 (TYR-2), and then generates indole-2-carboxylic acid-5,6-quinone (IQCA) under the action of tyrosinase-related protein 1 (TYR-1), and finally forms eumelanin.

[0074] The mechanism of action of whitening ingredients is mainly divided into five aspects from the cause, path and transfer of melanin:

[0075] (1) Inhibit the formation of melanin particles (inhibit tyrosinase activity, inhibit dopachrome isomerase activity, inhibit oxidation reaction);

[0076] (2) inhibiting the transfer of melanin granules to keratinocytes;

[0077] (3) Accelerate the transfer of melanin from keratinocytes to the stratum corneum and promote the shedding of the stratum corneum;

[0078] (4) Blocking the signal transduction pathway in the process of melanin production;

[0079] (5) Reduce the negative impact of exogenous factors such as ultraviolet rays on the melanin formation process.

[0080] From the perspective of melanin generation factors, in the rate-controlling stage of melanin formation, tyrosinase is the only rate-limiting enzyme in the reaction process, and its activity determines the amount of melanin synthesis. L-tyrosine in melanocytes is eventually converted into pigment substances under the action of tyrosinase. In this process, in addition to TYR, TRP-1, TRP-2 and peroxidase are also involved. In the process of melanin generation, whitening agents mainly reduce the production of melanin by inhibiting the activity of the above four enzymes or blocking the melanin oxidation pathway. Since in the process of melanin formation, the process of tyrosine synthesizing melanin under the action of tyrosinase requires the participation of oxygen, the generation of melanin can also be inhibited by scavenging oxygen free radicals, such as reducing exogenous stimuli such as oxygen free radicals and ultraviolet rays.

[0081] Tyrosinase is a key enzyme that is widely present in organisms. It plays an important role in melanin synthesis and other biological processes. It is a complex multi-subunit copper-containing oxidoreductase. Each subunit contains two metallic copper ions, which are covalently bound to the imino groups of three histidine residues and fixed on the active center, forming the active center of tyrosinase. It is widely present in microorganisms, animals, plants and humans, especially in the melanocytes of melanocytes.

[0082] Tyrosinase is the rate-limiting enzyme that regulates melanin production. It participates in two key reactions in melanin synthesis: one is the hydroxylation of monophenol into diphenol, and the other is the oxidation of o-diphenol into o-diquinone. After several steps of reaction, o-diquinone becomes melanin. Tyrosinase has a binuclear copper center structure. When substances such as tyrosine and enzymes are transitionally complexed, it is mainly the atomic bonding between the hydroxyl group and the active center of the enzyme that acts, thereby catalyzing the oxidation reaction of tyrosine to generate DOPA. DOPA is then converted into indolequinone through a series of oxidation, decarboxylation and other reactions, and finally indolequinone polymerizes to form melanin.

[0083] In the human body, tyrosinase is not only involved in the synthesis of melanin, but also affects physiological processes such as endocrine regulation and gene expression. Abnormal function or deficiency of tyrosinase may lead to skin pigmentation or loss, such as the occurrence of skin diseases such as vitiligo. Tyrosinase is also involved in physiological processes such as human metabolism, blood circulation, immunity enhancement, and lactose tolerance.

[0084] There is a close interactive relationship between anti-oxidation and anti-inflammation. Oxidative stress is caused by excessive free radicals or reactive oxygen species, which not only damages cells and tissues, but also activates inflammatory responses in the body. The generation of free radicals is often closely related to inflammatory processes such as the release of cytokines and the activation of leukocytes. Antioxidants neutralize free radicals and reactive oxygen species, reduce oxidative damage, and thus indirectly inhibit inflammatory responses. For example, antioxidants such as vitamin C, vitamin E, and phenolic compounds can remove excess free radicals in the body, reduce the production of inflammatory factors such as TNF-α and IL-6, and play an anti-inflammatory role. In addition, many plant extracts (such as green tea polyphenols, curcumin, etc.) have both antioxidant and anti-inflammatory effects, and can effectively reduce skin inflammatory responses caused by oxidative stress. Therefore, antioxidants and anti-inflammatory agents often work together through interaction to help maintain skin health. By enhancing the antioxidant capacity of the skin, antioxidants can effectively fight against the cellular mechanisms of aging, enhance the skin's self-repair ability, and slow down the aging process.

[0085] Natural product compounds have a significant effect on skin anti-inflammatory effects. Many plant extracts and natural compounds, such as aloe vera, polyphenols, flavonoids, green tea extracts, etc., have been shown to reduce skin inflammatory responses. For example, polysaccharides and flavonoids in aloe vera inhibit the release of inflammatory factors and reduce skin redness and irritation; catechins in green tea have antioxidant and anti-inflammatory effects and help relieve skin inflammation caused by ultraviolet rays. Flavonoids, such as quercetin, reduce skin allergic reactions by inhibiting the production of pro-inflammatory cytokines. In addition, natural products tend to have lower toxicity and are more suitable for use in skin care products. Therefore, natural product compounds provide a safe and effective alternative for skin anti-inflammatory treatment.

[0086] The raw materials and reagents used in the specific embodiments of the present invention are all commercially available products, and the purchase channels do not affect the realization of the technical effects.

[0087] Example 1

[0088] The preparation steps of the skin composition are as follows:

[0089] 5 mg of calycosin was weighed and dissolved in 560 μL of DMSO to prepare a skin composition with a concentration of 20 mM (about 0.8 wt %).

[0090] Test Example 1: ABTS method to detect total antioxidant capacity

[0091] Preparation of ABTS diamine salt stock solution: Weigh 38.5 mg of ABTS powder, add appropriate amount of ultrapure water to dissolve it completely, transfer it to a 10 mL volumetric flask and make up to volume to obtain 7 mM ABTS stock solution.

[0092] Preparation of K2S2O8 stock solution: Weigh 378 mg of K2S2O8 powder, add appropriate amount of ultrapure water to dissolve it completely and transfer it to a 10 mL volumetric flask to make up the volume to obtain 140 mM K2S2O8 stock solution.

[0093] Preparation of α-arbutin stock solution: Weigh 27.2 mg of α-arbutin and dissolve it in 5000 μL DMSO to prepare a 20 mM α-arbutin stock solution.

[0094] The 20 mM composition for skin prepared in Example 1 was diluted with ultrapure water to prepare working solutions of 80 μM, 40 μM, 20 μM, 10 μM, and 5 μM of calycosin glycosides.

[0095] The α-arbutin stock solution was diluted with ultrapure water to prepare α-arbutin working solutions with concentrations of 80 μM, 40 μM, 20 μM, 10 μM, and 5 μM.

[0096] Accurately pipette 5 mL of the above-mentioned 7 mM ABTS stock solution and 88 μL of the above-mentioned 140 mM K2S2O8 stock solution into a centrifuge tube, mix thoroughly and react for 16 h in the dark, then dilute with PBS buffer, and use a microplate reader to measure the absorbance at 734 nm until the absorbance value is about 0.7 to obtain ABTS working solution.

[0097] The reaction system was configured as shown in Table 1, and the mixture was thoroughly mixed by low-speed oscillation on an oscillator. After reacting at room temperature for 5 min, the absorbance was measured at 734 nm using an ELISA reader, and the free radical scavenging rate was calculated. The results are shown in Figure 1 shown.

[0098] Table 1

[0099]

[0100] Figure 1 This is the antioxidant performance diagram of glycosides of calycosin and α-arbutin detected by the ABTS method. It can be seen from the figure that at the concentrations of 5μM and 10μM, the free radical scavenging abilities of glycosides of calycosin and α-arbutin are similar. At the concentrations of 20μM, 40μM, and 80μM, the free radical scavenging ability of glycosides of calycosin is stronger than that of α-arbutin.

[0101] Test Example 2: FRAP method to detect total antioxidant capacity

[0102] The preparation of the working solution of calycosin isoflavone glycosides and α-arbutin is the same as that in Experimental Example 1.

[0103] Preparation of FRAP working solution: Use the FRAP method to measure total antioxidant capacity kit (Ragen), and prepare it at a ratio of FRAP Assay: TPTZ solution: ferric chloride solution = 10:1:1.

[0104] The reaction system was configured as shown in Table 2. After the reaction was kept at 37°C for 30 min, the absorbance was measured at 593 nm using an ELISA reader to calculate the free radical scavenging rate. The results are shown in Figure 2 shown.

[0105] Table 2

[0106]

[0107] Figure 2 This is the antioxidant performance diagram of calycosin and α-arbutin detected by FRAP method. It can be seen from the figure that at a concentration of 2.5-40μM, as the concentration of the active ingredient increases, the greater the absorbance value detected, the stronger the total antioxidant performance. The value shows that at the same concentration, the total antioxidant capacity of calycosin isoflavone glycosides is stronger than that of α-arbutin.

[0108] Test Example 3: Mushroom tyrosinase inhibition experiment

[0109] The preparation of α-arbutin working solution and calycosin working solution is the same as that in Experimental Example 1.

[0110] Use phosphate buffer to prepare 200 U / mL Agaricus bisporus tyrosinase solution and 2 mmol / L L-tyrosine solution.

[0111] The groups were set up, with three replicate wells in each group. The reaction was carried out on a 96-well cell plate. The reaction system was configured as shown in Table 3. The order of sample addition was tyrosinase solution, PBS buffer, and working solution (calycosin isoflavone glycoside working solution or α-arbutin working solution). After adding the samples in order, the temperature was kept constant at 37°C for 10 minutes, and then 2mmol / L L-tyrosine solution was added to each well. The well was then placed in an ELISA reader and reacted for 5 minutes (the concentration of calycosin isoflavone glycoside in the reaction system was 0-20μM). The OD value at 475nm was measured, and the inhibition rate was calculated. The results are shown in the figure. Figure 3 shown.

[0112] Table 3

[0113]

[0114] Figure 3 is the inhibition rate of α-arbutin and calycosin on mushroom tyrosinase, where a is α-arbutin and b is calycosin. It can be seen from the figure that calycosin has a significant inhibitory effect on mushroom-derived tyrosinase when the concentration is 5μM, and the inhibitory effect becomes stronger as the concentration increases. The highest concentration used in this test example is the concentration suitable for cell experiments. In the group with 20μM calycosin, the inhibition rate of mushroom tyrosinase is close to 25%, while α-arbutin has no significant inhibitory effect on mushroom tyrosinase.

[0115] Test example 4:

[0116] 4.1 Effects of DMSO on the proliferation and viability of B16F10 cells

[0117] Before culturing B16F10 cells, the effect of DMSO on the proliferation and viability of B16F10 cells was detected. The DMSO detection concentrations were set to 0.008%, 0.04%, 0.2%, and 1%.

[0118] B16F10 cells were collected, counted and diluted to 3 × 10 4 100 μL of each cell was inoculated into a 96-well plate and incubated overnight in a cell culture incubator to allow the cells to adhere completely.

[0119] After the cells were completely attached to the wall, the culture medium in the wells was aspirated, and the blank group cell culture medium was added in turn according to the experimental groups. The culture medium of each concentration of DMSO was cultured for 48 hours, and the proliferation and vitality of B16F10 cells were detected using the CCK8 detection kit. The results are as follows Figure 4 shown.

[0120] Figure 4 The results of the effect of DMSO on the viability of B16F10 cells show that DMSO content below 0.2% has no effect on B16F10 cell viability, while 1% concentration significantly affects cell viability. Therefore, in subsequent experiments, the amount of DMSO added when culturing B16F10 cells does not exceed 0.2%.

[0121] 4.2 Effects of calycosin and α-arbutin on the proliferation and viability of B16F10 cells

[0122] (1) Weigh 5 mg of calycosin and dissolve it in 560 μL of DMSO to prepare a calycosin stock solution with a concentration of 20 mM.

[0123] The above-mentioned calycosin isoflavone glycosides mother solution was diluted with DMEM high-glucose culture medium to prepare calycosin isoflavone glycosides working solutions with concentrations of 80 μM, 40 μM, 20 μM, 10 μM and 5 μM.

[0124] (2) Weigh 27.2 mg of α-arbutin and dissolve it in 5000 μL of DMSO to prepare a 20 mM α-arbutin stock solution.

[0125] The α-arbutin mother solution was diluted with DMEM high-glucose medium to prepare α-arbutin working solutions with concentrations of 80 μM, 40 μM, 20 μM, 10 μM, and 5 μM.

[0126] (3) Collect B16F10 cells, count and dilute to 5×10 4100 μL of each cell was inoculated into a 96-well plate and incubated overnight in a cell culture incubator to allow the cells to fully adhere to the wall.

[0127] After the cells were completely attached to the wall, the culture medium in the wells was aspirated, and the blank group cell culture medium (DMEM complete culture medium containing 10% FBS, 100 μL), the control group α-arbutin working solution (100 μL), and the experimental group calycosin working solution (100 μL) were added in turn according to the experimental groups and cultured for 48 hours. The CCK8 detection kit was used to detect the effect of calycosin on the proliferation and vitality of B16F10. The results are shown in Figure 2. Figure 5 shown.

[0128] Figure 5 The results of the effects of α-arbutin and calycosin on the proliferation and vitality of B16F10 cells, where (a) is α-arbutin and (b) is calycosin. As can be seen from the figure, among the concentrations of α-arbutin detected, the concentrations of 5μM and 10μM have no obvious toxicity to cells. When the drug concentration is 20μM, it has a weak toxicity to B16F10 cells, reducing the cell survival rate, but it is still 94.8%. The experimental results show that the concentrations of calycosin are 5μM, 10μ and 20μM. There is no obvious toxicity to cells. Therefore, three concentrations of 5μM, 10μM and 20μM were selected for subsequent experiments.

[0129] 4.3 Effects of calycosin on the expression of tyrosinase-related proteins

[0130] According to the results of 4.2, the concentrations of calycosin isoflavone glycosides were grouped into 5μM, 10μM, and 20μM, α-arbutin was set as the positive control, and the concentration of the positive control group was set to 20μM. After the cells were cultured in the medium containing the above concentrations of drugs for 48h, the cell protein was extracted for protein concentration quantification and the loading amount was unified. 15μg protein sample was loaded on each well, and Western-blot was performed to detect the expression level of intracellular tyrosinase, with β-actin as the internal reference. The results are as follows Figure 6 shown.

[0131] The culture medium is DMEM complete culture medium containing 10% FBS.

[0132] Figure 6 The figure shows the inhibition of tyrosinase expression in B16F10 cells by 100 μM α-MSH. At the same time, the tyrosinase expression in the experimental group was significantly reduced by adding 100 μM α-MSH.

[0133] 4.4. Effect of calycosin on the expression of β-galactosidase in photoaged cells:

[0134] When the fibroblast confluence reached 70-80%, the cells were collected by trypsin digestion and cell counting, and the cell concentration was adjusted to 1.0×10 6 The cells were inoculated into cell culture dishes at a density of 100 μg / dish, and then cultured in an incubator after adding complete medium. A blank group, a light group, and a drug-added group (20 μM calycosin) were set up.

[0135] Observe under the microscope when the cell fusion in the culture dish reaches 70%, aspirate the culture medium and wash three times with PBS. Add an appropriate amount of PBS to the dish to completely cover the floor of the dish, fix the UVB light source 15 cm away from the cells for irradiation, and use a UVB detector to detect the irradiation intensity during the irradiation process to determine the irradiation dose. After irradiation, aspirate the PBS and continue to culture according to the grouped culture medium. Irradiation is required for 5 consecutive days, once a day. After the irradiation cycle, aspirate the original culture medium in the dish, add different concentrations of drug-containing culture medium prepared with complete culture medium containing 10% FBS, and continue to culture for 48 hours before subsequent testing.

[0136] Use the cell senescence β-galactosidase staining kit according to the instructions to stain the β-galactosidase in the cells, and then randomly select the field of view under an optical microscope for observation and photography.

[0137] The experimental results are as follows Figure 7 As shown in the figure, the number of blue-green cells in the field of view in the light-treated group increased significantly and the color was darker, indicating that the β-galactosidase of HFF cells was active and the cells aged after continuous UVB irradiation. The number of blue-green cells in the field of view in the drug-treated group decreased and the color was slightly lighter, indicating that calycosin alleviated the photoaging damage caused by UVB to HFF cells and had anti-aging potential.

[0138] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0139] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A skin composition, characterized in that: The active ingredient of the skin composition includes calycosin.

2. The skin composition according to claim 1, wherein The skin composition also includes a solvent as a component.

3. The skin composition according to claim 2, characterized in that The solvent includes one or more of phosphate buffered saline, dimethyl sulfoxide, water, physiological saline, propylene glycol, polyethylene glycol, glycerol or vegetable oil.

4. The skin composition according to claim 2, wherein The concentration of the calycosin in the skin composition is 0.1 to 20 wt.%.

5. Use of the skin composition according to any one of claims 1 to 4 in daily chemical products.

6. Application of calycosin as an effective ingredient in whitening and skin care cosmetics.

7. Use of calycosin in cosmetics for inhibiting cellular melanin production.

8. Use of calycosin in inhibiting the expression of tyrosinase and in active cosmetics.

9. Application of calycosin in anti-inflammatory and antioxidant cosmetics.

10. Application of calycosin in anti-wrinkle and anti-aging cosmetics.

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