A whitening composition comprising ginseng and pomegranate and use thereof

Abstract

The present invention belongs to the field of cosmetic technology, and specifically relates to a whitening composition comprising ginseng and pomegranate and its use. The whitening composition comprises the following components: pomegranate, ginseng, 2-deoxy-4-epi-gallanthin and fusarium sclerotin. Experimental results show that the composition of the present invention has a significant inhibitory effect on tyrosinase activity and tyrosine-related protein expression, 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 that inhibit melanin synthesis or products that improve skin pigmentation, and has broad market prospects.

Description

Technical Field

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

[0002] With the rapid development of society and economy, people's demand for whitening products has gradually increased. More and more consumers tend to buy beauty products containing active ingredients from 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 skin color. When the skin is exposed to strong ultraviolet radiation, ultraviolet radiation can induce the production of oxygen free radicals, promote the expression of tyrosinase, and stimulate the local skin to produce melanin, resulting in skin pigmentation. However, long-term exposure to ultraviolet rays, as well as factors such as 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 facial beauty, but also cause certain mental stress and psychological problems for patients.

[0004] To reduce melanin production in the skin, the search for safe and effective bioactive substances with whitening properties has become increasingly important. Tyrosinase (TYR) is an oxidoreductase widely found in plants and animals and is a key enzyme in the biosynthesis of melanin. Melanin biosynthesis is a process catalyzed by tyrosinase from l-tyrosine to DOPA, which is further oxidized to DOPAQUINONE. DOPAQUINONE then undergoes a series of chemical reactions to produce melanin. Studies have shown that tyrosinase activity is correlated with melanin synthesis. Controlling tyrosinase activity can control melanin production. Inhibiting tyrosinase activity can reduce melanin production in the skin, thereby achieving skin whitening. Due to its role in skin pigmentation and the browning reaction during fruit harvesting and processing, tyrosinase inhibition has been a long-standing 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 are usually toxic. 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 known for its ability to inhibit melanosome transport, can be applied to the skin. The most commonly used whitening injections use kojic acid, tranexamic acid, or arbutin as active ingredients, achieving whitening by fundamentally inhibiting melanin synthesis. Due to their immediate whitening effects, whitening injections have been highly sought after by celebrities and beauty enthusiasts since their release. However, whitening injections are currently subject to significant controversy, with many professionals believing they pose significant safety risks. This suggests that the active ingredients used in current topical and injectable whitening products are unsatisfactory, and they all suffer from various safety and effectiveness deficiencies.

[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 existing bioactive substances that can inhibit tyrosinase activity, but the effects of individual active substances are often insignificant. Building on previous reports, the present invention discovered that a combination of four active ingredients, based on a combination of pomegranate and ginseng, can inhibit tyrosinase activity, thereby achieving skin whitening effects.

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

[0011] In a first aspect, the present invention provides a whitening composition of natural origin, comprising the following components: pomegranate, ginseng, 2-Desoxy-4-epi-pulchellin (CAS: 122872-03-1) and rubrofusarin (CAS: 3567-00-8).

[0012] As an optional mode, in the above whitening composition, the whitening composition is made of the following components: pomegranate, ginseng, 2-deoxy-4-epi-gaillarin and fusarium erythrin.

[0013] As an option, in the above whitening composition, the concentration of pomegranate is in the range of 0.5-1.2 μg / mL, the concentration of ginseng is in the range of 1-10 μg / mL, the concentration of 2-deoxy-4-epi-gaillarin is in the range of 0.1-1 μM, and the concentration of fusarium erythrin is in the range of 0.1-1 μM.

[0014] As an option, in the whitening composition, the concentration of pomegranate is 0.8 μg / mL, the concentration of ginseng is 4 μg / mL, the concentration of 2-deoxy-4-epi-gaillarin is 0.3 μM, and the concentration of fusarium erythrosine is 0.3 μM.

[0015] As an option, in the above whitening composition, pomegranate is pomegranate extract, and ginseng is ginseng extract.

[0016] Preferably, the pomegranate extract and / or ginseng extract are commercially available products.

[0017] As an optional manner, in the above whitening composition, various active ingredients in the whitening composition synergistically inhibit tyrosinase activity and inhibit tyrosine-related protein expression.

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

[0019] As an option, in the above use, the skin pigmentation is irregular skin pigmentation.

[0020] 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.

[0021] As an option, in the above use, the product is a cosmetic.

[0022] As an optional mode, in the above use, the cosmetic is a facial mask, a facial cream, an essence, an emulsion, a toner or a makeup.

[0023] As an optional mode, in the above-mentioned use, the amount of the whitening composition in the product is 0.5%-20% by weight.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (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.

[0026] (2) The whitening composition of the present invention can be used to prepare various skin whitening products or products that inhibit melanin synthesis or products that improve skin pigmentation, and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : Effects of each monomer and combination on the survival rate of B16-F10 cells.

[0028] Figure 2 : The inhibition rate of each monomer and combination on tyrosinase activity.

[0029] Figure 3 Effects of various monomers and combinations on TYR protein expression in B16-F10 cells. Left panel: WB bands; right panel: TYR protein expression analysis. DETAILED DESCRIPTION

[0030] 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents of this specification shall prevail.

[0033] 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 without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] 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 illustrate the present invention and are not intended to limit the scope of the present invention.

[0036] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0037] 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.

[0038] Example:

[0039] 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 melanin synthesis. Therefore, the expression and function of TYR are crucial in studies of melanoma and other melanin-related diseases. Studies have shown that overexpression of TYR in B16-F10 cells indicates a greater capacity for melanin synthesis, making them an ideal model for studying melanin production and its regulation. α-MSH-induced signaling pathways can regulate TYR activity. This process involves the binding of MC1R (melanocortin receptor 1) to α-MSH, which in turn influences cAMP (cyclic adenosine monophosphate) production and protein kinase A (PKA) activation, ultimately affecting TYR expression and function. This property provides a theoretical basis for studying melanin production, its regulatory mechanisms, and related diseases.

[0040] The data in this example were processed using GraphPad Prism 9.3.0 statistical software.

[0041] 1. Experimental Reagents

[0042] (1) Mouse melanoma cells B16-F10 were purchased from China Punosai Life Science Co., Ltd.

[0043] (2) Roswell Park Memorial Institute (RPMI) 1640 culture medium and FBS fetal bovine serum were purchased from Gibco, USA.

[0044] (3) Cell Counting Kit (CCK-8) cell proliferation-toxicity detection kit was purchased from Dojindo Chemical Research Institute, Japan.

[0045] (4) The tyrosinase activity detection kit was purchased from the official website of Merck Group in China.

[0046] (5) 2-Deoxy-4-epi-gaillarin (CAS: 122872-03-1) and fusarium erythrosin (CAS: 3567-00-8) were purchased from Tianjin WuXi AppTec New Drug Development Co., Ltd.

[0047] (6) Ginseng was a commercial ginseng extract purchased from Guangdong Qingyunshan Pharmaceutical Co., Ltd. and pomegranate was a commercial pomegranate extract purchased from Ningxia Vanilla Biotechnology Co., Ltd.

[0048] (7) All other chemical reagents are conventional reagents.

[0049] 2. Experimental Methods

[0050] 2.1 CCK-8 cytotoxicity assay

[0051] The CCK-8 assay was used to screen 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 prepared into a cell suspension using RPMI 1640 complete medium containing 10% fetal bovine serum and 1% double antibody. The cell suspension was then inoculated into a 96-well plate, with 100 μL of cell suspension in each well and 1×10 cells per well. 4 / well, culture overnight in a constant temperature incubator at 37°C and 5% CO2. When the cell confluence is about 80%, different concentrations of monomers of pomegranate, ginseng, 2-deoxy-4-epi-gaillarin (CAS: 122872-03-1), and fusarium mycelialin (CAS: 3567-00-8) are added, and the maximum concentration that has no obvious inhibitory effect on cell survival is selected for compounding.

[0052] The blank group was set as the culture medium only, the control group was set as the culture medium and cells without the test substance, and the experimental group was set as the culture medium and cells with the test substance. 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. The OD value of each well was measured at a wavelength of 450nm, and the proliferation and survival rate of the cells affected 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 each monomer and combination on the survival rate of B16-F10 cells are shown in the figure. Figure 1 .

[0053] The results showed that none of the tested monomers or compound concentrations had an inhibitory effect on B16-F10 cell viability. Therefore, subsequent tyrosinase activity inhibition and immunoblotting (WB) experiments were conducted using 0.8 μg / mL pomegranate, 4 μg / mL ginseng, 0.3 μM 2-deoxy-4-epi-gaillarin (CAS: 122872-03-1), 0.3 μM fusarium erythrin (CAS: 3567-00-8), and compound compounds.

[0054] 2.2 Evaluation of the inhibitory effects of monomers and combinations on tyrosinase activity

[0055] The above monomers and compositions and a positive control inhibitor (kojic acid) were selected to evaluate the inhibitory effect of the test substances on tyrosinase activity.

[0056] Method: Centrifuge the kojic acid / tyrosinase / tyrosinase substrate mixture at 12,000 rpm for 1 minute according to the Sigma MAK257 kit instructions. Prepare a 0.75 mM working solution of kojic acid in sterile water and keep on ice until ready to use. Dissolve the tyrosinase and tyrosinase substrate in 220 μL of tyrosinase assay buffer and 220 μL of sterile water, respectively, and keep on ice until ready to use.

[0057] (1) In a 96-well plate, add 20 μL of 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.

[0058] (2) Add 50 μL of enzyme system (48 μL tyrosinase assay buffer + 2 μL tyrosinase) to each well, mix well by pipetting, and let stand for 10 min.

[0059] (3) Add 30 μL of substrate system (23 μL tyrosinase assay buffer + 2 μL tyrosinase substrate + 5 μL tyrosinase enhancer) to each well, mix well by pipetting, and let stand for 30 min.

[0060] (4) Measure the absorbance at 510 nm using a microplate reader in kinetic mode for 30-60 minutes. Select two time points (T1 and T2) within the linear range and obtain the corresponding absorbance values ​​(Abs1 and Abs2). Calculate the slope (slope) = (Abs1 and Abs2) / (T1 and T2). Detect the inhibitory effects of the monomer and combination on tyrosinase activity and calculate the % relative inhibition rate as follows:

[0061] %Relative inhibition rate = [Slope (对照) –Slope (待测物 / 曲酸) ] / Slope (对照) ×100, the result is Figure 2 shown.

[0062] 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 is more effective than the monomer in inhibiting skin melanin synthesis.

[0063] 2.3 Evaluation of the effects of monomers and combinations on TYR protein expression in B16-F10 cells

[0064] The above monomers and compositions were used 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.

[0065] Methods: B16-F10 cells in logarithmic growth phase were taken, digested and prepared into 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 minutes, lysed on ice to extract total cell protein, and the protein concentration was determined by 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. Incubated overnight in a shaker at 4°C, the membrane was washed with TBS-T washing solution, HRP-labeled secondary antibody was added and incubated at room temperature for 1 hour, the membrane was washed with TBS-T washing solution, and photographed with a chemiluminescence imaging system. The results are shown in the figure. Figure 3 As shown, the left picture is WB band, and the right picture is the analysis of TYR protein expression level.

[0066] The results showed that after intervention with monomers and the 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.

[0067] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. Use of 2-deoxy-4-epi-gallarialin in the preparation of a product having an inhibitory effect on tyrosinase activity, characterized in that: The concentration of 2-deoxy-4-epi-gallophyline was 0.3 μM.

2. Use of the composition in preparing a product for inhibiting the expression of tyrosine-related proteins, characterized in that: The composition is prepared from the following components: pomegranate, ginseng, 2-deoxy-4-epi-gaillarin and fusarium erythrophyllin, wherein the concentration of pomegranate is 0.8 μg / mL, the concentration of ginseng is 4 μg / mL, the concentration of 2-deoxy-4-epi-gaillarin is 0.3 μM, and the concentration of fusarium erythrophyllin is 0.3 μM. The pomegranate is a commercial pomegranate extract, and the ginseng is a commercial ginseng extract.

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