Whitening composition containing American ginseng and folium mori and application of whitening composition

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a whitening composition containing American ginseng and folium mori and application of the whitening composition. The whitening composition is prepared from the following components: folium mori, radix panacis quinquefolii, (-)-Podophyllotoxin and (+)-Glaucarubinone, and is characterized in that the folium mori is used as a raw material; experimental results show that the composition has an obvious inhibiting effect on tyrosinase activity and tyrosine related protein expression, and then the effect of whitening the skin is achieved. Therefore, the whitening composition disclosed by the invention can be used for preparing various skin whitening products or products for inhibiting melanin synthesis or products for improving skin pigmentation, and has a wide market prospect.

Description

Technical Field

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

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

[0003] Melanin is a biological pigment found in both animals and plants. It is produced and stored by melanocytes. The content and distribution of melanin affect the skin color. When the skin is exposed to strong ultraviolet rays, ultraviolet rays induce the production of oxygen free radicals, promote the expression of tyrosinase, and stimulate the local skin to produce melanin, thus leading to skin pigmentation. However, long-term exposure to ultraviolet rays, increased stress in life, irregular work and rest schedules, or the use of inferior cosmetics can lead to an imbalance in the ability of human melanocytes to synthesize melanin, resulting in various pigmentation disorders, such as sun spots, chloasma, etc. Pigmentation disorders not only seriously affect the beauty of the face, but also bring certain mental stress and psychological problems to patients.

[0004] In order to reduce the production of melanin in the skin, it is more important to explore safe and effective bioactive substances with whitening effects. Tyrosinase (TYR) is an oxidoreductase that is widely present in plants and animals and is a key enzyme for the synthesis of melanin in organisms. The biosynthesis of melanin is a process in which tyrosinase catalyzes the conversion of l-tyrosine in the body into dopa, which is further oxidized to form dopaquinone, which undergoes a series of chemical reactions and finally forms melanin. Studies have shown that the activity of tyrosinase is related to the amount of melanin synthesis. Controlling its activity can control the amount of melanin production. Inhibiting tyrosinase activity can reduce the production of melanin in the skin, thereby achieving the purpose of skin whitening. Due to the role of tyrosinase in skin pigmentation and browning reactions during fruit harvesting and processing, the inhibition of tyrosinase has been a long-term goal in skin care research, cosmetics and agriculture.

[0005] Currently, the application routes of skin whitening products mainly include topical application, oral administration and whitening injections, and the active ingredients are mostly derived from chemically synthesized monomers, which usually have certain toxicity. When used alone, the whitening effect is poor. If the dosage is increased, it may cause certain toxic side effects to the human body, and the cost is relatively high.

[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: 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).

[0011] 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.

[0012] 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.

[0013] As an optional mode, in the above whitening composition, the concentration of mulberry leaf 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.

[0014] As an optional mode, in the above whitening composition, mulberry leaf is mulberry leaf extract, and American ginseng is American ginseng extract.

[0015] Preferably, the mulberry leaf extract and / or American ginseng extract are commercially available products.

[0016] As an optional mode, in the above whitening composition, various active ingredients in the whitening composition synergistically inhibit tyrosinase activity and inhibit tyrosine-related protein expression.

[0017] In a second aspect, the present invention provides use of the whitening composition described in the first aspect in preparing a skin whitening product or a product for inhibiting melanin synthesis or a product for improving skin pigmentation.

[0018] As an optional mode, in the above use, the skin pigmentation is irregular skin pigmentation.

[0019] As an optional mode, in the above use, the irregular skin pigmentation is one or more of age spots, freckles, dark spots, chloasma, sun spots or butterfly spots.

[0020] As an option, in the above use, the product is a cosmetic.

[0021] As an optional mode, in the above use, the cosmetic is a facial mask, a facial cream, an essence, a lotion, a toner or a makeup.

[0022] As an optional mode, in the above-mentioned use, the amount of the whitening composition in the product is 0.5%-20% by weight.

[0023] Compared with the prior art, the present invention has the following beneficial effects: (1) The present inventors screened and compounded a large number of active ingredients with whitening effects from natural sources, and obtained a whitening composition that can synergistically inhibit tyrosinase activity and tyrosine-related protein expression without obvious toxic side effects.

[0024] (2) 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Effects of each monomer and combination on the survival rate of B16-F10 cells.

[0026] Figure 2 : The inhibition rate of each monomer and combination on tyrosinase activity.

[0027] Figure 3 :Effects of each monomer and combination on TYR protein expression in B16-F10 cells. The left figure shows WB bands; the right figure shows the analysis of TYR protein expression levels.

[0028] Figure 4 : The results of human skin patch tests on two test substances. The left side of the left figure is the control group and the right side is the CAS: 518-28-5 group, and the left side of the right figure is the control group and the right side is the CAS: 1259-86-5 group. DETAILED DESCRIPTION

[0029] 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.

[0030] It should be understood that the terms described in the present invention are only for describing special embodiments 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. Each smaller range between the intermediate value in any stated value or stated range and 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.

[0031] 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.

[0032] 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 application description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] The present invention will be further described 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 intended to limit the scope of the present invention.

[0035] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0036] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0037] Example: B16-F10 cells are a commonly used melanoma cell line and are widely used in research to study the formation of melanin and related biological processes. Tyrosinase (TYR) is a rate-limiting enzyme involved in the synthesis of melanin. Therefore, in studies related to melanin production such as melanoma, the expression and function of TYR are important research objects. Studies have shown that B16-F10 cells overexpress TYR, which means that these cells have a stronger ability to synthesize melanin, making this cell an ideal model for studying melanin production and regulatory mechanisms. The α-MSH-induced signaling pathway can regulate the activity of TYR. This process involves the binding of MC1R (melanocortin receptor 1) to α-MSH, which in turn affects the generation 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 the production and regulatory mechanisms of melanin and related diseases.

[0038] The data processing in this example was performed using GraphPad Prism 9.3.0 statistical software.

[0039] 1. Experimental Reagents (1) Mouse melanoma cells B16-F10 were purchased from China Prosperity Life Science Co., Ltd.

[0040] (2) Roswell Park Memorial Institute (RPMI) 1640 culture medium and FBS fetal bovine serum were purchased from Gibco, USA.

[0041] (3) Cell Counting Kit (CCK-8) was purchased from Dojindo Chemical Research Institute, Japan.

[0042] (4) The tyrosinase activity detection kit was purchased from the official website of Merck Group in China.

[0043] (5) (-)-Podophyllotoxin (CAS: 518-28-5) and (+)-Glaucarubinone (CAS: 1259-86-5) were purchased from Tianjin WuXi AppTec Pharmaceutical Development Co., Ltd.

[0044] (6) American ginseng was a commercial American ginseng extract purchased from Guangdong Qingyunshan Pharmaceutical Co., Ltd., and mulberry leaf was a commercial mulberry leaf extract purchased from Bozhong Shengjing Pharmaceutical Technology (Beijing) Co., Ltd.

[0045] (7) Other chemical reagents are conventional reagents.

[0046] 2. Experimental Methods 2.1 CCK-8 cytotoxicity assay The CCK-8 method was used to screen out the concentration that had no significant inhibitory effect on the viability of B16-F10 cells. The method was as follows: B16-F10 cells in the logarithmic growth phase were taken, digested, and made into a cell suspension using RPMI 1640 complete medium containing 10% fetal bovine serum and 1% double antibody, and inoculated into a 96-well plate. 100 μL of cell suspension was inoculated in each well, and the number of cells was 1×10 4 / well, 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 concentration that had no obvious inhibitory effect on cell survival rate was selected for compounding.

[0047] The group with only culture medium was set as the blank group, the culture medium and cells without the test substance were set as the control group, and the culture medium and cells with the test substance were set as the experimental group. The cells treated with the test substance were placed in a 37°C, 5% CO2 constant temperature incubator for 12 hours. After the original culture medium was replaced with a culture medium containing 10% CCK-8, the 96-well plate was placed in a constant temperature incubator for 4 hours, and the OD value of each well was measured at a wavelength of 450 nm. The proliferation survival rate of cells by different test substances 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The results of the effects of various monomers and combinations on the survival rate of B16-F10 cells are shown in Figure 1 .

[0048] The results showed that the concentrations of the monomers and compound test substances that had been determined had no inhibitory effect on the survival rate of B16-F10 cells. Therefore, the monomers 0.8 μg / mL mulberry leaves, 5 μg / mL American ginseng, 0.3 μM (-)-Podophyllotoxin (CAS: 518-28-5), 0.3 μM (+)-Glaucarubinone (CAS: 1259-86-5) and compound test substances were used for subsequent tyrosinase activity inhibition rate experiments and Western blot (WB) experiments.

[0049] 2.2 Evaluation of the inhibitory effects of monomers and combinations on tyrosinase activity The above monomers and combinations and a positive control inhibitor (kojic acid) were selected to evaluate the inhibitory effect of the test substance on tyrosinase activity.

[0050] Method: According to the instructions of the Sigma MAK257 kit, centrifuge kojic acid / tyrosinase / tyrosinase substrate at 12000rpm for 1 min. Prepare kojic acid into a 0.75 mM working solution with sterile water and place it on ice for later use. Dissolve tyrosinase and tyrosinase substrate with 220 μL tyrosinase assay buffer and 220 μL sterile water respectively and place them on ice for later use.

[0051] (1) In a 96-well plate, add 20 μL of the test drug 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.

[0052] (2) Add 50 μL of enzyme system (48 μL tyrosinase assay buffer + 2 μL tyrosinase) to each well, pipette to mix well, and let stand for 10 min.

[0053] (3) Add 30 μL of substrate system (23 μL tyrosinase assay buffer + 2 μL tyrosinase substrate + 5 μL tyrosinase enhancer) to each well, mix by pipetting, and let stand for 30 min.

[0054] (4) Use a microplate reader to measure the absorbance value at 510 nm in kinetic mode for a period of 30-60 minutes. 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 and Abs2) / (T1 and T2). Detect the inhibitory effect of the monomer and the combination on tyrosinase activity, and calculate the % relative inhibition rate as follows: %Relative inhibition rate = [Slope (对照) –Slope (待测物 / 曲酸) ] / Slope (对照) ×100, the result is Figure 2 shown.

[0055] The results showed that after adding the monomer and the combination, the combination had a more significant inhibitory effect on tyrosinase activity than the monomer, indicating that the combination was more effective than the monomer in inhibiting the synthesis of skin melanin.

[0056] 2.3 Evaluation of the effects of monomers and combinations on TYR protein expression in B16-F10 cells The above monomers and combinations were selected to treat B16-F10 cells, and Western blot experiments were performed after protein extraction to evaluate the expression level of TYR protein in B16-F10 cells.

[0057] The method is as follows: B16-F10 cells in the logarithmic growth phase were taken, digested, and made into a cell suspension using RPMI 1640 complete medium containing 10% fetal bovine serum and 1% double antibody, and 7×10 6 The cells were cultured in 6 cm cell culture dishes, 5 mL per dish, in a constant temperature incubator at 37°C and 5% CO2. After 24 hours of adaptive culture, the control group was replaced with fresh complete culture medium, and the test intervention group was replaced with fresh complete culture medium containing the above-mentioned monomers and compositions. After 24 hours of culture in a constant temperature incubator at 37°C and 5% CO2, the cells were scraped off with a cell spatula, centrifuged at 1000 rpm for 5 min, lysed on ice to extract total cell protein, and the protein concentration was determined by the BCA kit; 30 μg of protein was taken from each group for SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked with 5% BSA, and TYR and GAPDH primary antibodies were added. Incubate overnight in a shaker at 4°C, wash the membrane with TBS-T washing solution, add HRP-labeled secondary antibody and incubate at room temperature for 1 hour, wash the membrane with TBS-T washing solution, and take pictures with a chemiluminescence imaging system. The results are as follows Figure 3 As shown, the left figure is WB band, and the right figure is the analysis of TYR protein expression level.

[0058] The results showed that after intervention with the monomer and combination, the expression level of TYR protein in B16-F10 cells was significantly reduced, and the effect of the combination was significantly better than that of the monomer.

[0059] 2.4 Human skin patch test Human skin patch test, usage method is based on: "Cosmetic Safety Technical Specifications" (2015 edition). General test method: Select a suitable patch tester, use the closed patch test method, select qualified volunteers to participate in the test, and add the test substance, distilled water (deionized water) and control sample into the square chamber of the patch tester. Apply the patch tester with the test substance, distilled water (deionized water) and control sample to the inner side of the subject's forearm, and press it lightly with the palm of your hand to evenly apply it to the skin for 24 hours. Observe the skin reaction 30 minutes (after the indentation disappears) and 8 hours after removing the patch tester with the test substance, distilled water (deionized water) and control sample, and record the observation results.

[0060] The results are as follows Figure 4 As shown, the left side of the left figure is the control group and the right side is the CAS: 518-28-5 group, and the left side of the right figure is the control group and the right side is the CAS: 1259-86-5 group. The results show that both test substances have no obvious irritation to the skin.

[0061] 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 their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A whitening composition of natural origin, characterized in that: The whitening composition comprises the following components: mulberry leaves, American ginseng, (-)-Podophyllotoxin and (+)-Glaucarubinone.

2. The whitening composition according to claim 1, characterized in that: The whitening composition is prepared from the following components: mulberry leaves, American ginseng, (-)-Podophyllotoxin and (+)-Glaucarubinone.

3. The whitening composition according to claim 1, characterized in that: In the 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.

4. The whitening composition according to claim 1, characterized in that: Various active ingredients in the whitening composition synergistically inhibit tyrosinase activity and tyrosine-related protein expression.

5. Use of the whitening composition according to any one of claims 1 to 4 in the preparation of a skin whitening product, a product for inhibiting melanin synthesis, or a product for improving skin pigmentation.

6. The use according to claim 5, characterized in that: The skin pigmentation is irregular skin pigmentation.

7. The use according to claim 6, characterized in that: The irregular skin pigmentation is one or more of age spots, freckles, dark spots, chloasma, sun spots or butterfly spots.

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

9. The use according to claim 8, characterized in that: The cosmetics are facial masks, facial creams, essences, lotions, toners or makeup.

10. The use according to claim 5, characterized in that: In terms of weight percentage, the amount of the whitening composition in the product is 0.5%-20%.

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