A whitening composition containing American ginseng and mulberry leaves and its uses

Abstract

The present invention belongs to the technical field of cosmetics, and particularly relates to a whitening composition containing American ginseng and mulberry leaves and its uses. The whitening composition comprises the following components: mulberry leaves, American ginseng, (-)-Podophyllotoxin, and (+)-Glaucarubinone. Experimental results show that the composition of the present invention has obvious inhibitory effects on both tyrosinase activity and the expression of tyrosine-related proteins, thereby achieving the effect of skin whitening. Therefore, the whitening composition of the present invention can be used to prepare various skin whitening products, or products for inhibiting melanin synthesis, or products for improving skin pigmentation, and has broad market prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and particularly relates to a whitening composition containing American ginseng and mulberry leaves and its uses. Background Art

[0002] With the rapid development of social economy, people's demand for whitening products has gradually increased. More and more consumers tend to buy beauty products containing active ingredients of natural products, which poses higher requirements and challenges for the development of skin beauty products.

[0003] Melanin is a biological pigment contained in both animals and plants, which is generated and stored by melanocytes. The content and distribution of melanin affect skin color. When the skin is strongly irradiated by ultraviolet rays, ultraviolet rays can induce the generation of oxygen free radicals, promote the expression of tyrosinase, and stimulate the local skin to generate melanin, thus leading to skin pigmentation. However, long-term exposure to ultraviolet rays, as well as factors such as increased life pressure, irregular work and rest, or the use of inferior cosmetics, can all cause the disorder of the ability of human melanocytes to synthesize melanin, resulting in various abnormal pigment depositions, such as solar lentigines, melasma, etc. Pigment disorders not only seriously affect facial beauty but also bring certain mental stress and psychological problems to patients.

[0004] In order to reduce the generation of skin melanin, it becomes more important to explore safe and effective bioactive substances with whitening effects. Tyrosinase (TYR) is an oxidoreductase widely present in plants and animals and is a key enzyme for the biosynthesis of melanin in organisms. The biosynthesis of melanin is a process in which tyrosinase catalyzes the generation of dopa from l-tyrosine in the body, dopa is further oxidized to dopaquinone, and dopaquinone undergoes a series of chemical reactions to finally generate melanin. Research shows that the activity of tyrosinase is related to the amount of melanin synthesis. Controlling its activity can control the amount of melanin generation. Inhibiting the activity of tyrosinase can reduce the generation of skin melanin, and thus achieve the purpose of skin whitening. Due to the role of tyrosinase in skin pigmentation and the browning reaction during fruit harvesting and processing, the inhibition of tyrosinase has always been a long-term goal in the fields of skin health research, cosmetics, and agriculture.

[0005] Currently, the application methods of current skin whitening products mainly include topical application, oral administration, and whitening injections, and most of the active ingredients are derived from chemically synthesized monomer substances, which usually have certain toxicity, have poor whitening effects when used alone, may cause certain toxic side effects to the human body if the dosage is increased, and have high costs.

[0006] For example, niacinamide, a representative ingredient that inhibits the transport of melanosomes, can be applied to the skin. The most commonly used whitening injections use kojic acid, tranexamic acid or arbutin as active ingredients, which achieve the purpose of whitening by fundamentally inhibiting the synthesis of melanin. Since the whitening effect of whitening injections is immediate, they have been sought after by beauty lovers as soon as they were launched on the market. However, whitening injections are currently very controversial, and many professionals believe that they have very high safety risks. It can be seen that the active ingredients used in current topical and injectable whitening products are not satisfactory, and they have more or less defects in safety and effectiveness.

[0007] Therefore, the development of new safe and efficient whitening products has significant application and commercial value. Summary of the invention

[0008] In order to solve the problem of insufficient whitening products of natural origin in the prior art, the present invention uses active ingredients of natural origin for compounding to prepare a whitening composition of natural origin with low cost, wide source, good whitening effect and no toxic side effects.

[0009] In particular, there are many biologically active substances that can inhibit the activity of tyrosinase, but the effect of a single active substance is often not obvious. Based on the original report, the present invention found that the combination of four active ingredients based on mulberry leaves and American ginseng has the effect of inhibiting the activity of tyrosinase, thereby achieving the effect of skin whitening.

[0010] Specifically, the present invention is realized through the following technical solutions:

[0011] In a first aspect, the present invention provides a whitening composition of natural origin, comprising the following components: mulberry leaf, American ginseng, (-)-Podophyllotoxin (CAS: 518-28-5) and (+)-Glaucarubinone (CAS: 1259-86-5).

[0012] As an optional mode, in the above whitening composition, the whitening composition is made of the following components: mulberry leaves, American ginseng, (-)-Podophyllotoxin and (+)-Glaucarubinone.

[0013] As an option, in the above whitening composition, the concentration range of mulberry leaf is 0.5-1.2 μg / mL, the concentration range of American ginseng is 1-10 μg / mL, the concentration range of (-)-Podophyllotoxin is 0.1-1 μM, and the concentration range of (+)-Glaucarubinone is 0.1-1 μM.

[0014] As an alternative, in the above-mentioned whitening composition, the concentration of mulberry leaves is 0.8 μg / mL, the concentration of American ginseng is 5 μg / mL, the concentration of (-)-Podophyllotoxin is 0.3 μM, and the concentration of (+)-Glaucarubinone is 0.3 μM.

[0015] As an alternative, in the above-mentioned whitening composition, the mulberry leaves are mulberry leaf extract, and the American ginseng is American ginseng extract.

[0016] Preferably, the mulberry leaf extract and / or the American ginseng extract are commercially available products obtained by purchase.

[0017] As an alternative, in the above-mentioned whitening composition, the various active ingredients in the whitening composition synergistically inhibit tyrosinase activity and inhibit the expression of tyrosine-related proteins.

[0018] In a second aspect, the present invention provides the use of the whitening composition described in the first aspect above in the preparation of a skin whitening product or a product for inhibiting melanin synthesis or a product for improving skin pigmentation.

[0019] As an alternative, in the above-mentioned use, the skin pigmentation is irregular skin pigmentation.

[0020] As an alternative, in the above-mentioned use, the irregular skin pigmentation is one or more of senile plaques, freckles, dark spots, melasma, sunburns or butterfly spots.

[0021] As an alternative, in the above-mentioned use, the product is a cosmetic.

[0022] As an alternative, in the above-mentioned use, the cosmetic is a facial mask, a cream, a serum, a lotion, a toner or makeup.

[0023] As an alternative, in the above-mentioned use, calculated by weight percentage, the dosage of the whitening composition in the product is 0.5% - 20%.

[0024] The present invention has the following beneficial effects compared with the prior art:

[0025] (1) After screening and compounding a large number of active ingredients with whitening effects from natural sources, the inventor of the present invention obtained a whitening composition that can synergistically inhibit tyrosinase activity and the expression of tyrosine-related proteins and has no obvious toxic and side effects.

[0026] (2) The whitening composition of the present invention can be used in the preparation of various skin whitening products or products for inhibiting melanin synthesis or products for improving skin pigmentation, and has broad market prospects. Description of the Drawings

[0027] Figure 1 : Effects of each monomer and composition on the viability of B16-F10 cells.

[0028] Figure 2 : Inhibition rates of each monomer and composition on tyrosinase activity.

[0029] Figure 3 : Effects of each monomer and composition on the expression of TYR protein in B16-F10 cells. Among them, the left figure is the WB band; the right figure is the analysis of the expression level of TYR protein.

[0030] Figure 4 : Results of human skin patch tests for two test substances. Among them, on the left side of the left figure is the control group and on the right side is the group of CAS: 518-28-5, and on the left side of the right figure is the control group and on the right side is the group of CAS: 1259-86-5. Detailed implementation manners

[0031] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0034] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of this application are only exemplary.

[0035] Regarding the terms "comprising", "including", "having", "containing", etc. used in this article, they are all open-ended terms, meaning including but not limited to.

[0036] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the scope of the present invention.

[0037] For those not specifying specific techniques or conditions in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0038] In the following examples, the experimental methods are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

[0039] Example:

[0040] B16-F10 cells are a commonly used melanoma cell line and are widely used in research to study melanin formation and related biological processes. Tyrosinase (TYR) is a rate-limiting enzyme involved in the synthesis process of melanin. Therefore, in research related to melanin production such as melanoma, the expression and function of TYR are important research objects. Research shows that B16-F10 cells overexpress TYR, which means these cells have a stronger ability to synthesize melanin, making these cells an ideal model for studying melanin production and regulatory mechanisms. The signal pathway induced by α-MSH can regulate the activity of TYR. This process involves the binding of MC1R (melanocortin receptor 1) to α-MSH, which in turn affects the production of cAMP (cyclic adenosine monophosphate) and the activation of protein kinase A (PKA), ultimately affecting the expression and function of TYR. This characteristic provides a theoretical basis for studying melanin production, regulatory mechanisms, and related diseases.

[0041] For the data processing in this example, GraphPad Prism 9.3.0 statistical software was used to process the data.

[0042] 1. Experimental reagents

[0043] (1) Mouse melanoma cells B16-F10 were purchased from Wuhan Procell Life Science & Technology Co., Ltd.

[0044] (2) Roswell Park Memorial Institute (RPMI) 1640 medium and fetal bovine serum (FBS) were purchased from Gibco, USA.

[0045] (3) The Cell Counting Kit (CCK-8) for cell proliferation and toxicity detection was purchased from Dojindo Laboratories, Japan.

[0046] (4) The tyrosinase activity detection kit was purchased from the official website of Merck Group China.

[0047] (5) (-)-Podophyllotoxin (CAS: 518-28-5) and (+)-Glaucarubinone (CAS: 1259-86-5) were purchased from WuXi AppTec Co., Ltd., Tianjin.

[0048] (6) American ginseng was a commercialized American ginseng extract purchased from Guangdong Qingyunshan Pharmaceutical Co., Ltd., and mulberry leaf was a commercialized mulberry leaf extract purchased from Bozhong Shengjing Medical Technology (Beijing) Co., Ltd.

[0049] (7) All other chemical reagents were conventional reagents.

[0050] 2. Experimental methods

[0051] 2.1 CCK-8 cytotoxicity experiment

[0052] The CCK-8 method was used to screen out the concentration that had no obvious inhibitory effect on the viability of B16-F10 cells. The method was as follows: B16-F10 cells in the logarithmic growth phase were digested and made into a cell suspension with RPMI 1640 complete medium containing 10% fetal bovine serum and 1% double antibody. The cell suspension was inoculated into a 96-well plate, 100 μL of the cell suspension was inoculated into each well and the cell number was 1×10 4 / well. It was cultured overnight in a constant temperature incubator at 37°C and 5% CO2. When the cell confluence was about 80%, different concentrations of monomers of mulberry leaf, American ginseng, (-)-Podophyllotoxin (CAS: 518-28-5) and (+)-Glaucarubinone (CAS: 1259-86-5) were added, and the concentrations that had no obvious inhibitory effect on cell viability were selected for compounding of the composition.

[0053] The group with only the medium was set as the blank group, the medium and cells without the test substance were set as the control group, and the medium and cells containing the test substance were set as the experimental group. The cells treated with the test substance were placed in a constant temperature incubator at 37°C and 5% CO2 and cultured for another 12 h. After replacing the original medium with the medium containing 10% CCK-8, the 96-well plate was placed in the constant temperature incubator for 4 h, and the OD value of each well was measured at a wavelength of 450 nm. The proliferation survival rate of different test substances on cells was calculated according to the formula. Proliferation survival rate (%) = { (OD 实验组 - OD 空白组 ) / (OD 对照组 - OD空白组 ))} × 100%. The results of the viability of B16-F10 cells for each monomer and composition are as Figure 1 .

[0054] The results showed that the concentrations of the measured monomers and the complex test substances had no inhibitory effect on the viability of B16-F10 cells. Therefore, the monomers 0.8 μg / mL mulberry leaf, 5 μg / mL American ginseng, 0.3 μM (-)-Podophyllotoxin (CAS: 518-28-5), 0.3 μM (+)-Glaucarubinone (CAS: 1259-86-5) and the complex were used for the subsequent tyrosinase activity inhibition rate experiment and immunoblot (Western blot, WB) experiment.

[0055] 2.2 Evaluation of the inhibitory effect of monomers and combinations on tyrosinase activity

[0056] The above-mentioned monomers, compositions and the positive control inhibitor (kojic acid) were selected to evaluate the inhibitory effect of the test substances on tyrosinase activity.

[0057] The method was as follows: Kojic acid / tyrosinase / tyrosinase substrate was centrifuged at 12,000 rpm for 1 min according to the Sigma MAK257 kit instructions. Kojic acid was prepared as a 0.75 mM working solution with sterile water and placed on ice for later use. Tyrosinase and tyrosinase substrate were dissolved in 220 μL of tyrosinase detection buffer and 220 μL of sterile water respectively and placed on ice for later use.

[0058] (1) In a 96-well plate, 20 μL of the test drug was added successively as the experimental well, 20 μL of 0.75 mM kojic acid as the positive control well, and 20 μL of tyrosinase assay buffer as the negative control well.

[0059] (2) 50 μL of the enzyme-containing system (48 μL of tyrosinase assay buffer + 2 μL of tyrosinase) was added to each well, pipetted and mixed well, and left standing for 10 min.

[0060] (3) 30 μL of the substrate system (23 μL of tyrosinase assay buffer + 2 μL of tyrosinase substrate + 5 μL of tyrosinase enhancer) was added to each well, pipetted and mixed well, and left standing for 30 min.

[0061] (4)Measure the absorbance values in the time period of 30 - 60 minutes at 510 nm in kinetic mode using a microplate reader. Select two time points (T1 and T2) within the linear range and obtain the corresponding absorbance values (Abs1 and Abs2), and calculate the slope = (Abs1 - Abs2) / (T1 - T2). Detect the inhibitory effects of the monomer and the composition on tyrosinase activity, and calculate the % relative inhibition rate as follows:

[0062] % relative inhibition rate = [Slope (对照) –Slope (待测物 / 曲酸) / Slope (对照) × 100, and the results are as Figure 2 shown.

[0063] The results showed that after adding the monomer and the composition, the inhibitory effect of the composition on tyrosinase activity was more obvious than that of the monomer. It indicates that the composition has a better effect on inhibiting skin melanin synthesis than the monomer.

[0064] 2.3 Evaluate the effects of the monomer and the composition on the expression of TYR protein in B16-F10 cells

[0065] Select the above monomer and composition to treat B16-F10 cells. After extracting the protein, perform Western blot experiments to evaluate the expression level of TYR protein in B16-F10 cells.

[0066] The method is as follows: Take B16-F10 cells in the logarithmic growth phase, digest them and make a cell suspension with RPMI 1640 complete medium containing 10% fetal bovine serum and 1% double antibody. Inoculate 7×10 6 into a 6 cm cell culture dish, 5 mL per dish, and culture in a constant temperature incubator at 37℃ and 5% CO2. After 24 h of adaptive culture, the control group is replaced with fresh complete medium, and the test substance intervention group is replaced with fresh complete medium containing the above monomer and composition. After culturing in a constant temperature incubator at 37℃ and 5% CO2 for 24 h, scrape the cells with a cell scraper, centrifuge at 1000 rpm for 5 min, lyse and extract the total cell protein on ice, and measure the protein concentration with a BCA kit; Take 30 μg of protein from each group for SDS-PAGE electrophoresis, transfer to a PVDF membrane, block with 5% BSA, and add primary antibodies against TYR and GAPDH. Incubate overnight on a shaker at 4℃, wash the membrane with TBS-T washing solution, add HRP-labeled secondary antibody and incubate at room temperature for 1 h, wash the membrane with TBS-T washing solution, and take pictures with a chemiluminescence imaging system. The results are as Figure 3 shown, where the left figure is the WB band and the right figure is the analysis of the expression level of TYR protein.

[0067] The results showed that after the intervention of the monomer and the composition, the expression level of TYR protein in B16-F10 cells decreased significantly, and the effect of the composition was significantly better than that of the monomer.

[0068] 2.4 Human skin patch test

[0069] For the human skin patch test, the usage method is based on the "Technical Specifications for Cosmetics Safety" (2015 edition). General test method: Select a suitable patch test tester, and use the closed patch test method. Select qualified volunteers to participate in the test. Add the test substance, distilled water (deionized water), and control sample into the square chamber of the patch test tester. Apply the patch test tester with the test substance, distilled water (deionized water), and control sample on the inner side of the forearm of the subject, and gently press with the palm to make it evenly adhere to the skin for 24 h. Observe the skin reaction 30 min (after the indentation disappears) and 8 h after removing the patch test tester with the test substance, distilled water (deionized water), and control sample, and record the observation results.

[0070] The results are as Figure 4 shown. The left side of the left figure is the control group and the right side is the CAS: 518-28-5 group. The left side of the right figure is the control group and the right side is the CAS: 1259-86-5 group. The results indicate that neither of the two test substances has obvious irritating effects on the skin.

[0071] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and its equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. A whitening composition of natural origin, characterized in that: The whitening composition is made from the following components: mulberry leaf extract, American ginseng extract, (-)-Podophyllotoxin, and (+)-Glaucarubinone. In the whitening composition, the concentration of the mulberry leaf extract is 0.8 μg / mL, the concentration of the American ginseng extract is 5 μg / mL, the concentration of (-)-Podophyllotoxin is 0.3 μM, and the concentration of (+)-Glaucarubinone is 0.3 μM. The various active ingredients in the whitening composition synergistically reduce the expression of tyrosinase (TYR) protein.

2. Use of the whitening composition according to claim 1 in the preparation of a skin whitening product, or a product for inhibiting melanin synthesis, or a product for improving skin pigmentation.

3. The use according to claim 2, characterized in that: The skin pigmentation is irregular skin pigmentation.

4. Use according to claim 3, characterized in that: The irregular skin pigmentation is one or more of senile plaques, freckles, dark spots, melasma, sunburn, or butterfly spots.

5. The use according to claim 2, characterized in that: The product is a cosmetic.

6. The use according to claim 5, characterized in that: The cosmetic is a facial mask, facial cream, essence, lotion, toner, or makeup.

7. The use according to claim 2, wherein: Calculated by weight percentage, the dosage of the whitening composition in the product is 0.5% - 20%.

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