Whitening composition containing panacis quinquefolii radix and mori folium, and use thereof

Abstract

A whitening composition containing the following components: Mori folium, Panacis quinquefolii radix, (-)-podophyllotoxin, and (+)-glaucarubinone. Further provided is the use of the whitening composition in the preparation of a product for whitening the skin, or a product for inhibiting melanin synthesis, or a product for improving skin pigmentation.

Description

A skin-whitening composition comprising American ginseng and mulberry leaves and its uses

[0001] Related applications

[0002] This application claims priority to Chinese patent application filed on January 7, 2025, application number 202510022361.4, entitled "A whitening composition comprising American ginseng and mulberry leaves and its use thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application belongs to the field of cosmetic technology and relates to a whitening composition comprising American ginseng and mulberry leaves and its uses. Background Technology

[0004] With the rapid development of society and the economy, people's demand for skin whitening products is gradually increasing. More and more consumers tend to buy beauty products containing natural active ingredients, which poses higher requirements and challenges for the development of skin beauty products.

[0005] Melanin is a biological pigment found in both plants and animals. It is produced and stored by melanocytes, and its content and distribution affect skin color. When skin is exposed to strong ultraviolet (UV) radiation, UV rays induce the production of oxygen free radicals, promoting the expression of tyrosinase and stimulating local skin melanin production, thus leading to skin pigmentation. However, prolonged exposure to UV radiation, increased life stress, irregular sleep patterns, or the use of inferior cosmetics can all disrupt the body's ability to synthesize melanin, causing various pigmentation abnormalities, such as sunspots and melasma. Pigmentation abnormalities not only severely affect facial appearance but also cause significant mental stress and psychological problems for patients.

[0006] To reduce melanin production in the skin, the search for safe and effective bioactive substances with skin-whitening effects has become increasingly important. Tyrosinase (TYR) is a redox enzyme widely found in plants and animals, and is a key enzyme in the synthesis of melanin. Melanin biosynthesis involves tyrosinase catalyzing the production of dopa from L-tyrosine in the body. Dopa is further oxidized to dopaquinone, which then undergoes a series of chemical reactions to finally produce melanin. Studies have shown that tyrosinase activity is related to the amount of melanin synthesized; controlling its activity can control the amount of melanin produced. Inhibiting tyrosinase activity can reduce melanin production in the skin, thereby achieving 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 health research, cosmetics, and agriculture.

[0007] Currently, skin whitening products are mainly applied topically, orally, and through whitening injections. Most of their active ingredients are derived from chemically synthesized monomers, which often have a certain degree of toxicity. When used alone, they have a poor whitening effect. If the dosage is increased, they may cause certain toxic side effects to the human body, and the cost is also high.

[0008] For example, niacinamide, a representative ingredient that inhibits melanosome transport, can be applied topically. Whitening injections most commonly use kojic acid, tranexamic acid, or arbutin as active ingredients, achieving whitening by fundamentally inhibiting melanin synthesis. Because whitening injections produce immediate whitening effects, they have been highly sought after by many celebrities and beauty enthusiasts since their introduction to the market. However, whitening injections are currently highly controversial, with many professionals believing they pose significant safety risks. It is evident that the active ingredients used in current topical and injectable whitening products are not entirely satisfactory, and they all have varying degrees of deficiencies in safety and effectiveness.

[0009] Therefore, developing new, safe, and effective skin whitening products has significant application and commercial value. Summary of the Invention

[0010] To address the shortage of naturally derived skin whitening products in traditional technologies, various embodiments of this application utilize naturally derived active ingredients in combination to prepare a low-cost, widely available, effective, and non-toxic skin whitening composition.

[0011] In particular, while there are various bioactive substances that can inhibit tyrosinase activity, the effects of a single active substance are often not significant. Based on previous reports, this application discovers that a combination of four active ingredients, based on mulberry leaf and American ginseng, inhibits tyrosinase activity, thereby achieving a skin whitening effect.

[0012] In some implementations, this application is achieved through the following technical solutions:

[0013] In a first aspect, this application provides a skin whitening composition derived from natural sources, the skin whitening composition comprising the following components: mulberry leaf, American ginseng, (-)-Podophyllotoxin (CAS: 518-28-5) and (+)-Glaucarubinone (CAS: 1259-86-5).

[0014] In some embodiments, the whitening composition described above is made from the following components: mulberry leaf, American ginseng, (-)-Podophyllotoxin, and (+)-Glaucarubinone.

[0015] In some embodiments, the concentration range of mulberry leaf in the above-mentioned whitening composition 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.

[0016] In some embodiments, the whitening composition contains mulberry leaf at a concentration of 0.8 μg / mL, American ginseng at a concentration of 5 μg / mL, (-)-Podophyllotoxin at a concentration of 0.3 μM, and (+)-Glaucarubinone at a concentration of 0.3 μM.

[0017] In some embodiments, in the above-mentioned whitening composition, mulberry leaf is mulberry leaf extract and American ginseng is American ginseng extract.

[0018] In some implementations, mulberry leaf extract and / or American ginseng extract are commercially available products that are purchased.

[0019] In some embodiments, in the above-described whitening composition, the various active ingredients in the whitening composition synergistically inhibit tyrosinase activity and inhibit the expression of tyrosine-related proteins.

[0020] In a second aspect, this application provides the use of the whitening composition described in the first aspect above in the preparation of skin whitening products, products that inhibit melanin synthesis, or products that improve skin pigmentation.

[0021] In some embodiments, in the above-described uses, the skin pigmentation is irregular skin pigmentation.

[0022] In some embodiments, in the above-described uses, the irregular skin pigmentation is one or more of age spots, freckles, dark spots, melasma, sun spots, or butterfly spots.

[0023] In some embodiments, in the above-described uses, the product is a cosmetic.

[0024] In some embodiments, in the above-described uses, the cosmetic is a face mask, face cream, serum, lotion, toner, or makeup.

[0025] In some embodiments, in the above-described uses, the amount of the whitening composition in the product is 0.5%-20% by weight.

[0026] Details of one or more embodiments of the present invention are set forth in the following drawings and description. Other features, objects, and advantages of the invention will become apparent from the specification, drawings, and claims. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0028] Figure 1 shows the effects of various monomers and compositions on the survival rate of B16-F10 cells;

[0029] Figure 2 shows the inhibition rate of each monomer and composition on tyrosinase activity;

[0030] Figure 3 shows the effects of various monomers and compositions on TYR protein expression in B16-F10 cells. The left panel shows Western blotting bands; the right panel shows the analysis of TYR protein expression levels.

[0031] Figure 4 shows the results of human skin patch tests for the two test substances. In the left figure, the left side represents the control group and the right side represents the CAS: 518-28-5 group, and in the right figure, the left side represents the control group and the right side represents the CAS: 1259-86-5 group. Detailed Implementation

[0032] Various exemplary embodiments of this application are now described in detail. This detailed description should not be considered as a limitation of this application, but rather as a more detailed description of certain aspects, features, and implementations of this application.

[0033] It should be understood that the terminology used in this application is merely for describing particular embodiments and is not intended to limit the application. Furthermore, for numerical ranges in this application, 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 stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this application. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art described herein. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this application. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this application without departing from the scope or spirit of this application, as will be apparent to those skilled in the art. Other embodiments derived from this application will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] The present application will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the present application.

[0038] In a first aspect, this application provides a skin whitening composition derived from natural sources, the skin whitening composition comprising the following components: mulberry leaf, American ginseng, (-)-Podophyllotoxin (CAS: 518-28-5) and (+)-Glaucarubinone (CAS: 1259-86-5).

[0039] The inventors of this application, through screening and compounding a large number of naturally derived active ingredients with whitening effects, have obtained a whitening composition that can synergistically inhibit tyrosinase activity and tyrosine-related protein expression without significant toxic side effects. The provided whitening composition can be used to prepare various skin whitening products, products that inhibit melanin synthesis, or products that improve skin pigmentation, and has broad market prospects.

[0040] In some embodiments, the whitening composition described above is made from the following components: mulberry leaf, American ginseng, (-)-Podophyllotoxin, and (+)-Glaucarubinone.

[0041] In some embodiments, the concentration range of mulberry leaf in the above-mentioned whitening composition 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.

[0042] Understandably, in the above-mentioned whitening composition, the concentration range of mulberry leaves can be any value between 0.5-1.2 μg / mL, for example, it can be 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, 1.0 μg / mL, 1.1 μg / mL, 1.2 μg / mL, etc., or it can be a range composed of any two of the aforementioned values.

[0043] Understandably, in the above-mentioned whitening composition, the concentration range of American ginseng can be any value between 1 and 10 μg / mL, for example, it can be 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, etc., or it can be a range composed of any two of the aforementioned values.

[0044] Understandably, in the above-mentioned whitening composition, the concentration range of (-)-Podophyllotoxin can be any value between 0.1 and 1 μM, for example, it can be 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, etc., or it can be a range composed of any two of the aforementioned values.

[0045] Understandably, in the above-mentioned whitening composition, the concentration range of (+)-Glaucarubinone can be any value between 0.1 and 1 μM, for example, it can be 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, etc., or it can be a range composed of any two of the aforementioned values.

[0046] In some embodiments, the whitening composition contains mulberry leaf at a concentration of 0.8 μg / mL, American ginseng at a concentration of 5 μg / mL, (-)-Podophyllotoxin at a concentration of 0.3 μM, and (+)-Glaucarubinone at a concentration of 0.3 μM.

[0047] In some embodiments, in the above-mentioned whitening composition, mulberry leaf is mulberry leaf extract and American ginseng is American ginseng extract.

[0048] In some implementations, mulberry leaf extract and / or American ginseng extract are commercially available products that are purchased.

[0049] In some embodiments, in the above-described whitening composition, the various active ingredients in the whitening composition synergistically inhibit tyrosinase activity and inhibit the expression of tyrosine-related proteins.

[0050] In a second aspect, this application provides the use of the whitening composition described in the first aspect above in the preparation of skin whitening products, products that inhibit melanin synthesis, or products that improve skin pigmentation.

[0051] In some embodiments, in the above-described uses, the skin pigmentation is irregular skin pigmentation.

[0052] In some embodiments, in the above-described uses, the irregular skin pigmentation is one or more of age spots, freckles, dark spots, melasma, sun spots, or butterfly spots.

[0053] In some embodiments, in the above-described uses, the product is a cosmetic.

[0054] In some embodiments, in the above-described uses, the cosmetic is a face mask, face cream, serum, lotion, toner, or makeup.

[0055] In some embodiments, in the above-described uses, the amount of the whitening composition in the product, by weight percentage, is 0.5%-20%, for example, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc., or may be a range of any two of the aforementioned values.

[0056] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0057] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0058] Example:

[0059] B16-F10 cells are a commonly used melanoma cell line, widely used in research to study melanin formation and related biological processes. Tyrosinase (TYR) is a rate-limiting enzyme involved in melanin synthesis; therefore, TYR expression and function are important research subjects in studies related to melanin production, such as melanoma. Studies have shown that overexpression of TYR in B16-F10 cells indicates a stronger ability to synthesize melanin, making this cell type an ideal model for studying melanin production and regulatory mechanisms. The α-MSH-induced signaling pathway regulates TYR activity. This process involves the binding of MC1R (melanocortin receptor 1) to α-MSH, which in turn affects cAMP (cyclic adenosine monophosphate) production and protein kinase A (PKA) activation, ultimately influencing TYR expression and function. This characteristic provides a theoretical basis for studying melanin production, regulatory mechanisms, and related diseases.

[0060] In this embodiment, the data processing uses GraphPad Prism 9.3.0 statistical software to process the data.

[0061] 1. Experimental reagents

[0062] (1) Mouse melanoma cells B16-F10 were purchased from Pronosei Life Sciences Co., Ltd., China.

[0063] (2) Roswell Park Memorial Institute (RPMI) 1640 medium and FBS fetal bovine serum were purchased from Gibco, USA.

[0064] (3) The Cell Counting Kit (CCK-8) cell proliferation-toxicity assay kit was purchased from Dojin Chemical Research Institute, Japan.

[0065] (4) The tyrosinase activity assay kit was purchased from the Merck Group China website.

[0066] (5) (-)-Podophyllotoxin (CAS: 518-28-5) and (+)-Glaucarubinone (CAS: 1259-86-5) were purchased from Tianjin WuXi AppTec New Drug Development Co., Ltd.

[0067] (6) The American ginseng was a commercially available American ginseng extract purchased from Guangdong Qingyunshan Pharmaceutical Co., Ltd., and the mulberry leaf was a commercially available mulberry leaf extract purchased from Bozhong Shengjing Pharmaceutical Technology (Beijing) Co., Ltd.

[0068] (7) All other chemical reagents are conventional reagents.

[0069] 2. Experimental Methods

[0070] 2.1 CCK-8 Cytotoxicity Assay

[0071] The concentration that did not significantly inhibit the viability of B16-F10 cells was screened using the CCK-8 assay. The method was as follows: B16-F10 cells in the logarithmic growth phase were digested and then suspended in RPMI 1640 complete medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics. The suspension was then seeded into 96-well plates at a density of 100 μL per well, with a cell count of 1 × 10⁶ cells. 4 / well, cultured overnight in a constant temperature incubator at 37℃ 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. The concentrations that did not significantly inhibit cell viability were selected for compounding.

[0072] The following groups were set up: a blank group (containing only culture medium), a control group (containing culture medium and cells without the analyte), and an experimental group (containing culture medium and cells with the analyte). Cells treated with the analyte were incubated at 37°C with 5% CO2 for 12 hours. The original culture medium was then replaced with medium containing 10% CCK-8, and the 96-well plates were placed in an incubator for 4 hours. The OD value of each well was measured at 450 nm. The cell proliferation and survival rate of different analytes was calculated using the formula: Proliferation and survival rate (%) = {(OD value)} 实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组 The results of each monomer and composition on the survival rate of B16-F10 cells are shown in Figure 1.

[0073] The results showed that none of the monomers and compound analytes had an inhibitory effect on the survival rate of B16-F10 cells. Therefore, subsequent tyrosinase activity inhibition rate experiments and Western blot (WB) experiments were conducted using 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 compound analytes.

[0074] 2.2 Evaluation of the inhibitory effects of monomers and combinations on tyrosinase activity

[0075] The above monomers and compositions, along with a positive control inhibitor (kojic acid), were selected to evaluate the inhibitory effect of the test substance on tyrosinase activity.

[0076] The method was as follows: Following the instructions of the Sigma MAK257 kit, kojic acid / tyrosinase / tyrosinase substrate was centrifuged at 12000 rpm for 1 min. Kojic acid was dissolved in sterile water to prepare a 0.75 mM working solution and kept on ice. Tyrosinase and tyrosinase substrate were dissolved separately in 220 μL of tyrosinase detection buffer and 220 μL of sterile water, respectively, and kept on ice until needed.

[0077] (1) Add 20 μL of test drug as experimental well, 20 μL of 0.75 mM kojic acid as positive control well, and 20 μL of tyrosinase assay buffer as negative control well to a 96-well plate in sequence.

[0078] (2) Add 50 μL of enzyme system (48 μL tyrosinase assay buffer + 2 μL tyrosinase) to each well, mix by pipetting, and let stand for 10 min.

[0079] (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.

[0080] (4) Measure the absorbance over a period of 30-60 minutes using a microplate reader at 510 nm in kinetic mode. Select two time points (T1 and T2) within the linear range and obtain the corresponding absorbance values ​​(Abs1 and Abs2). Calculate the slope = (Abs1 and Abs2) / (T1 and T2). Inhibitory effects of the monomer and composition on tyrosinase activity were detected, and the % relative inhibition rate was calculated as follows:

[0081] % relative inhibition rate = [Slope] (对照) –Slope (待测物 / 曲酸) ] / Slope (对照) ×100, the result is shown in Figure 2.

[0082] The results showed that, after the addition of both the monomer and the composition, the composition exhibited a more significant inhibitory effect on tyrosinase activity than the monomer. This indicates that the composition is more effective than the monomer in inhibiting melanin synthesis in the skin.

[0083] 2.3 Evaluation of the effects of monomers and compositions on TYR protein expression in B16-F10 cells

[0084] The above monomers and compositions were used to treat B16-F10 cells. After protein extraction, Western blot experiments were performed to evaluate the expression level of TYR protein in B16-F10 cells.

[0085] The method was as follows: B16-F10 cells in the logarithmic growth phase were taken, digested, and then a cell suspension was prepared in RPMI 1640 complete medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibodies. The suspension was then inoculated at a rate of 7 × 10⁶ cells / year. 6 Cells were cultured in 6cm cell culture dishes (5 mL per dish) at 37℃ in a 5% CO2 incubator. After 24 hours of adaptation culture, the control group was replaced with fresh complete culture medium, while the test group was replaced with fresh complete culture medium containing the aforementioned monomers and compositions. After 24 hours of culture at 37℃ in a 5% CO2 incubator, cells were scraped off with a cell scraper, centrifuged at 1000 rpm for 5 min, lysed on ice, and total protein was extracted. Protein concentration was determined using a BCA kit. 30 μg of protein from each group was subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked with 5% BSA, and incubated with TYR and GAPDH primary antibodies. The membrane was incubated overnight at 4℃ on a shaker, washed with TBS-T washing buffer, incubated with HRP-labeled secondary antibody at room temperature for 1 hour, washed with TBS-T washing buffer again, and photographed using a chemiluminescence imaging system. The results are shown in Figure 3, where the left image shows Western blotting (WB) bands, and the right image shows the TYR protein expression level analysis.

[0086] The results showed that the expression level of TYR protein in B16-F10 cells was significantly reduced after intervention with monomers and the combination, and the effect of the combination was significantly better than that of the monomer.

[0087] 2.4 Human skin patch test

[0088] Human skin patch testing, the method of use is based on: *Cosmetic Safety Technical Specifications* (2015 edition). General testing method: Select a suitable patch tester and use a closed patch test method. Select qualified volunteers to participate in the test. Add the test substance, distilled water (deionized water), and control sample to the square chamber of the patch tester. Apply the patch tester containing the test substance, distilled water (deionized water), and control sample to the inner forearm of the subject, and gently press with the palm to ensure even application to the skin. Continue for 24 hours. Observe the skin reaction 30 minutes after removing the patch tester (after the indentation disappears) and 8 hours later, and record the results.

[0089] The results are shown in Figure 4. In the left figure, the left side represents the control group and the right side represents the CAS: 518-28-5 group, and in the right figure, the left side represents the control group and the right side represents the CAS: 1259-86-5 group. The results indicate that neither test substance had a significant irritant effect on the skin.

[0090] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. A skin whitening composition of natural origin, the skin whitening composition comprising the following components: mulberry leaf, American ginseng, (-)-Podophyllotoxin and (+)-Glaucarubinone.

2. The whitening composition according to claim 1, wherein, The whitening composition is made from the following components: mulberry leaf, American ginseng, (-)-Podophyllotoxin and (+)-Glaucarubinone.

3. The whitening composition according to claim 1, wherein, 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, wherein, The various active ingredients in the whitening composition synergistically inhibit tyrosinase activity and the expression of tyrosine-related proteins.

5. Use of the whitening composition according to any one of claims 1 to 4 in the preparation of skin whitening products, products that inhibit melanin synthesis, or products that improve skin pigmentation.

6. The use according to claim 5, wherein, The skin pigmentation is irregular.

7. The use according to claim 6, wherein, The irregular skin pigmentation is one or more of the following: age spots, freckles, dark spots, melasma, sun spots, or butterfly spots.

8. The use according to claim 5, wherein, The product in question is a cosmetic.

9. The use according to claim 8, wherein, The cosmetics mentioned are face masks, face creams, serums, lotions, toners, or makeup.

10. The use according to claim 5, wherein, The whitening composition is used in the product at a weight percentage of 0.5%-20%.

Images