Application of glabridin or glabridin derivative in melanin inhibition and inflammation resistance

Abstract

The invention discloses application of glabrione or a derivative thereof in melanin inhibition and inflammation resistance. According to the invention, the application of the glabridin or the derivative thereof in preparing the whitening, blackening and / or anti-inflammatory products is proposed for the first time, so that the glabridin or the derivative thereof replaces the existing other licorice active ingredients. Moreover, the invention also finds that the glabridin or the derivative thereof has far better effects in the aspects of fading black and resisting inflammation than the existing liquorice active component, and the glabridin or the derivative thereof is higher in safety and lower in cost, so that the glabridin or the derivative thereof has extremely high practical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and particularly to the application of glabrone or its derivatives in melanin inhibition and anti - inflammation. Background Art

[0002] Licorice is a perennial herb of the genus Glycyrrhiza in the legume family. In the field of traditional Chinese medicine, Glycyrrhiza glabra has a sweet taste and a neutral nature, and has the effects of invigorating the spleen and replenishing qi, relieving cough and resolving phlegm, clearing heat and detoxifying, and coordinating the properties of medicines. It is mainly used to treat symptoms such as spleen - stomach weakness, qi deficiency in the middle - jiao, cough and asthma, palpitations and shortness of breath, sore throat, carbuncles and sores. The roots and rhizomes of Glycyrrhiza glabra can be used as medicine, which can relieve eye inflammation, treat digestive system inflammation (oral ulcers, gastritis, peptic ulcers and hyperacidity), chest pain, arthritis and certain skin diseases, and can also be used as a laxative.

[0003] The roots of Glycyrrhiza glabra contain a large number of components such as flavonoids, isoflavonoids, triterpenoid saponins, polysaccharides, phytosterols, coumarins and asparagine. Among them, isoflavones belong to estrogens, and glycyrrhizin has the same anti - inflammatory and anti - arthritis effects as corticosteroids or other corticosteroid hormones, and can also stimulate the adrenal gland to produce hormones and slow down the degradation of hormones in the liver and kidneys. Glabridin is a kind of isoflavone in Glycyrrhiza glabra, and it is the main component that exerts the whitening effect in licorice. It is known as the "whitening gold" and has very good tyrosinase inhibitory activity. However, its content is only relatively high in Glycyrrhiza glabra L. Glabridin can bind well to the active pocket of tyrosinase, prevent the formation of the tyrosinase - L - tyrosine complex, thereby inhibiting the copper - ion - catalyzed oxidation reaction of tyrosinase, and finally inhibiting the production of melanin. However, in addition to the melanin inhibition through the tyrosinase pathway based on glabridin, there is little knowledge in the prior art about the whitening and melanin - inhibiting effects of other active components in licorice, nor is it clear whether there are effective active components that can reduce the production of melanin through other ways such as inhibiting tyrosine or dopa oxidation, reducing the expression of tyrosinase, or improving the accumulation of skin melanin by promoting melanin metabolism, such as scavenging and transporting.

[0004] In the prior art, small molecule compounds capable of inhibiting melanin production mainly include vitamin C, arbutin, kojic acid, etc. However, these small molecule compounds have many usage limitations or defects in practical applications. For example, the anti-melanin effect of vitamin C is mainly based on substituting substrate oxidation, but it itself does not have tyrosinase inhibitory effect and has the disadvantages of easy consumption and poor effect; kojic acid easily causes allergies or skin problems; arbutin has tyrosinase inhibitory effect but the inhibitory ability is weak, and it does not have other effects such as anti-inflammatory. Glabridin commonly used on the market has good tyrosinase inhibitory and anti-inflammatory effects, but it has high cytotoxicity, and the source is single, resulting in disadvantages such as high price and high synthesis cost.

[0005] Therefore, there is an urgent need to develop a new anti-inflammatory and whitening active ingredient. Summary of the Invention

[0006] The present invention aims to at least solve one of the above technical problems existing in the prior art. For this reason, the object of the present invention is to provide the use of glabrone or its derivatives in melanin inhibition and anti-inflammation. The present invention first proposes to use glabrone or its derivatives to prepare whitening and melatonin-reducing and / or anti-inflammatory products, thereby replacing other existing licorice active ingredients. Moreover, the present invention also discovers that the effects of glabrone or its derivatives in melatonin reduction and anti-inflammation are far superior to the existing licorice active ingredients, and have higher safety and lower cost. Therefore, it has extremely high practical application value.

[0007] The first aspect of the present invention provides the use of glabrone or its derivatives in the preparation of whitening and melatonin-reducing and / or anti-inflammatory products.

[0008] In the present invention, glabrone specifically refers to the compound shown in Formula I, and its chemical name is: compound 7-hydroxy-3-(5-hydroxy-2,2-dimethylchromen-6-yl)chromen-4-one, with the molecular formula C 20 H 16 O5, and the CAS number is 60008-02-8.

[0009]

[0010] Those skilled in the art can understand that the form of glabrone in the present invention is not limited, and can be various forms such as the active compound itself, free form, its salts, esters, isomers, optical isomers, stereoisomers, regioisomers, geometric isomers, hydrates, non-hydrates, solvates or non-solvates, amorphous, crystalline, pharmaceutically acceptable cocrystals or cocrystal salts, derivatives, prodrugs, etc.

[0011] In the present invention, the prodrug includes a compound that can be converted into the active ingredient in a living organism under physiological conditions due to reactions with enzymes, gastric acid, etc., that is, a compound that can be converted into the active ingredient through enzyme-catalyzed oxidation, reduction, hydrolysis, etc.; a compound that can be converted into the active ingredient through hydrolysis, etc. due to gastric acid, and so on.

[0012] In the present invention, a cocrystal or cocrystal salt refers to a crystalline substance composed of two or more specific substances. At room temperature, each substance is a solid and has different physical properties (e.g., structure, melting point, heat of fusion, hygroscopicity, solubility, stability, etc.). Cocrystals and cocrystal salts can be prepared using co-crystallization methods that are already known.

[0013] In some embodiments of the present invention, the derivatives include pharmaceutically acceptable salts, solvate compounds, tautomers, or mixtures thereof of glabrone.

[0014] In some embodiments of the present invention, when the derivative is a pharmaceutically acceptable salt, examples of such salts include metal salts, ammonium salts, salts formed with organic bases, salts formed with inorganic acids, salts formed with organic acids, salts formed with basic or acidic amino acids, and so on. Among them, examples of metal salts include: alkali metal salts, such as sodium salts, potassium salts, etc.; alkaline earth metal salts, such as calcium salts, magnesium salts, barium salts, etc.; and aluminum salts. Examples of salts formed with organic bases include salts formed with the following organic bases: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, and so on. Examples of salts formed with inorganic acids include: salts formed with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Examples of salts formed with organic acids include salts formed with the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and so on. Examples of salts formed with basic amino acids include salts formed with the following basic amino acids: arginine, lysine, ornithine, and so on. Examples of salts formed with acidic amino acids include salts formed with the following acidic amino acids: aspartic acid, glutamic acid, and so on.

[0015] In some embodiments of the present invention, the product includes pharmaceuticals and cosmetics.

[0016] In some embodiments of the present invention, the product does not contain ingredients that violate the relevant laws and regulations of pharmaceuticals or cosmetics, or does not contain ingredients that exceed the specified limits in the relevant laws and regulations of pharmaceuticals or cosmetics.

[0017] In some embodiments of the present invention, the product is an external-use product.

[0018] In some embodiments of the present invention, the topical product is a solution, suspension, powder, emulsion, ointment, paste, plaster, cream, gel, liniment, film-forming agent, spray, aerosol, patch or roll-on.

[0019] In some embodiments of the present invention, the topical product further includes tincture, lotion and oil.

[0020] In some embodiments of the present invention, the topical product is a gel, ointment, liniment or cream.

[0021] In some embodiments of the present invention, the topical product is a solution or roll-on.

[0022] In some embodiments of the present invention, the topical product contains a solvent.

[0023] In some embodiments of the present invention, the solvent includes conventional topical solvents in the art, including but not limited to at least one of water, ethanol, propylene glycol, glycerol, polyethylene glycol, isopropanol, n-butanol, pentanol, turpentine, liquid paraffin, petrolatum, fatty oil, ethyl acetate and triethanolamine.

[0024] In some embodiments of the present invention, in the product, the content of glabrone or its derivative is greater than or equal to 0.1 w / w%.

[0025] In some embodiments of the present invention, in the product, the content of glabrone or its derivative is 0.1 - 20 w / w%.

[0026] In some embodiments of the present invention, in the product, the content of glabrone or its derivative is 1 - 20 w / w%.

[0027] In some embodiments of the present invention, in the product, the content of glabrone or its derivative is 5 - 20 w / w%.

[0028] In some embodiments of the present invention, the product further contains adjuvants, and the adjuvants include pharmaceutically or cosmetically acceptable adjuvants.

[0029] In some embodiments of the present invention, the adjuvants include excipients or carriers.

[0030] In the present invention, the term "excipient and / or carrier" refers to a carrier and / or excipient that is compatible with the subject and the active agent pharmacologically and / or physiologically, and is well-known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19 th(ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to pH regulators, surfactants, adjuvants, and ionic strength enhancers. For example, pH regulators include but are not limited to phosphate buffers; surfactants include but are not limited to cationic, anionic, or nonionic surfactants, such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride.

[0031] In some embodiments of the present invention, the carrier refers to various organic or inorganic carrier substances commonly used as formulation raw materials, without particular limitation.

[0032] In some embodiments of the present invention, the adjuvants in the cosmetics and pharmaceuticals include but are not limited to at least one of antioxidants, preservatives, bactericides, moisturizers, emulsifiers, fragrances, or pigments.

[0033] In some embodiments of the present invention, the product further contains a second active ingredient.

[0034] In some embodiments of the present invention, the function of the second active ingredient is the same as or different from that of glabrone or its derivatives.

[0035] In the present invention, the substances with "the same function" refer to substances having similar or close efficacy to glabrone or its derivatives in terms of whitening and melatonin reduction and / or anti-inflammatory effects. The substances with "different functions" refer to substances that do not have or have insignificant whitening and melatonin reduction and / or anti-inflammatory effects.

[0036] In some embodiments of the present invention, the second active ingredient includes but is not limited to licorice extract, active ingredients in licorice, or a mixture thereof.

[0037] In some embodiments of the present invention, the whitening and melatonin reduction include: inhibiting melanin production and / or reducing tyrosinase activity.

[0038] In some embodiments of the present invention, the whitening and melatonin reduction may further include: reducing tyrosinase expression or production, or interfering with, inhibiting, or regulating the tyrosinase-dopa-melanin related pathway or pathway.

[0039] The beneficial effects of the present invention are:

[0040] (1) The present invention first discovers that glabrone can significantly inhibit cell melanin production and has obvious anti-inflammatory effects. Moreover, compared with the existing active compounds derived from licorice, glabrone has a stronger inhibitory effect on melanin production and anti-inflammatory effect than the existing compounds, and at the same time has lower cytotoxicity.

[0041] (2) Glabrone in the present invention has a wide source, a relatively simple structure, and low synthesis difficulty. It can achieve whitening through multiple channels, including entering the active site of tyrosinase to inhibit the process of its oxidizing L-tyrosine, inhibiting melanin production, or regulating the relevant signal pathways of cells and affecting the process of cell synthesizing melanin. Moreover, it can synchronously reduce the generation of ROS and peroxides in the body and reduce the occurrence of inflammation. It has extremely high application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Shows the inhibitory effects of different concentrations of glabrone and glabridin on intracellular melanin production.

[0043] Figure 2 Shows the effects of different concentrations of glabrone and glabridin on cell viability.

[0044] Figure 3 Shows the effects of different concentrations of glabrone and glabridin on the production of nitric oxide in cells.

[0045] Figure 4 Shows the inhibitory effects of different concentrations of glabrone and glabridin on tyrosinase activity. DETAILED DESCRIPTION OF THE INVENTION

[0046] The content of the present invention will be further described in detail through specific examples below. The raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial channels or can be obtained by existing technical methods without special instructions. Unless otherwise specified, the test or measurement methods are conventional methods in the art.

[0047] Example 1

[0048] In this example, mouse skin melanoma cells (B16-F10 cells) were used as experimental objects to test the inhibitory effect of glabrone on melanin. The specific experimental method is as follows.

[0049] Inoculate B16-F10 cells at about 150,000 cells / well in a 6-well plate, culture them with DMEM complete medium, and after 24 h of adherence, respectively give 0, 1, 10, and 50 μg / mL of glabrone, continue to culture for 48 h, then collect the cells and count. Take about 100,000 cells from each group and transfer them into a 1.5 mL centrifuge tube, centrifuge at 800×g for 10 minutes. The precipitate is then bathed in a water bath at 80 °C for 1 hour with 200 μL of 1 μM sodium hydroxide containing 10% DMSO to fully dissolve it, centrifuge, remove the precipitate, and take 50 μL of the solution to detect the absorbance at 490 nm on an enzyme-linked immunosorbent assay (ELISA) reader. Use the wells without cells as the blank group. Calculate the melanin inhibition rate using the following formula:

[0050]

[0051] According to the above method, the absorbance after applying liquiritin apioside at 0, 1, 10, and 50 μg / mL respectively was detected as a positive control.

[0052] The test results are as Figure 1 shown.

[0053] From Figure 1 the results shown, it can be seen that both liquiritin apioside and liquiritone can inhibit the production of intracellular melanin. Taking the model group (i.e., the 0 μg / mL group) as a reference (100 ± 3.0%), at the doses of 1 and 10 μg / mL of liquiritin apioside, the production amount of melanin was reduced to 56.9 ± 3.0% and 15.6 ± 2.9% respectively, while for liquiritone, they were 35.2 ± 2.1% and 5.9 ± 1.1% respectively. It can be seen from this that liquiritone has a better inhibitory effect on melanin production than liquiritin apioside (**P<0.01).

[0054] Example 2

[0055] In this example, mouse skin melanoma cells (B16-F10 cells) were used as experimental objects to test the effect of liquiritone on cell viability. The specific experimental method is as follows.

[0056] B16-F10 cells were seeded in 6-well plates at about 5000 cells / well and cultured using DMEM complete medium. After 24 h of adhesion, 0, 1, 10, and 50 μg / mL of liquiritone were added respectively. After continuing to culture for 48 h, the drug-containing medium was discarded, and DMEM basal medium containing 0.5 mg / mL of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) was added, and the culture was continued in an incubator for 4 h. Then the medium containing MTT was discarded, 150 μg / mL / well of DMSO was added, and after shaking at room temperature for 5 min, 50 μL of the solution was taken to detect the absorbance at 490 nm on an enzyme-linked immunosorbent assay (ELISA) reader. The wells without cells were used as the blank group. The cell viability was calculated using the following formula:

[0057]

[0058] According to the above method, the absorbance after applying liquiritin apioside at 0, 1, 10, and 50 μg / mL respectively was detected as a control.

[0059] The test results are as Figure 2 shown.

[0060] From Figure 2As can be seen from the results shown, glabridin has stronger cytotoxicity to B16-F10 than glabrone, especially at the concentrations of 25 and 50 μg / mL. The cell viability of the cells treated with glabridin is significantly lower than that of the cells treated with glabrone (P<0.01), indicating that glabridin has greater cytotoxicity to B16-F10 cells. Thus, glabrone is superior to glabridin in terms of safety.

[0061] Example 3

[0062] In this example, the anti-inflammatory effect of glabrone was tested using mouse monocyte macrophage leukemia cells (Raw264.7 cells) as the experimental subjects, and the specific experimental method is as follows.

[0063] Raw264.7 cells were seeded in 96-well plates at about 50,000 cells / well and cultured using DMEM complete medium. After 24 h of adhesion, the medium was replaced with DMEM complete medium containing 0.1 mg / mL LPS and continued to be cultured. At the same time, glabrone at 0, 1, 10, and 100 μg / mL was respectively added, and after continuing to culture for 48 h, 50 μL of the culture supernatant was taken, and a nitric oxide detection kit (S0021S, Beyotime) was used to detect the nitric oxide concentration in the culture medium according to the instructions.

[0064] According to the above method, the nitric oxide concentrations after respectively adding glabridin at 0, 1, 10, and 100 μg / mL were detected as a control at the same time.

[0065] The detection results are as Figure 3 shown.

[0066] From Figure 3 As can be seen from the results shown, in the case of no treatment, the concentration of nitric oxide produced by LPS-induced Raw264.7 macrophages in the medium was 18.9±1.2 μM. Glabridin at the dosing doses of 1, 10, and 100 μg / mL could all reduce the production amount of nitric oxide, and its detected amounts in the medium were 12.7±0.6 μM, 2.9±1.1 μM, and 2.3±0.2 μM respectively. In contrast, at the same concentrations, the detected amounts of nitric oxide in the groups administered glabrone were 10.0±0.5 μM, 3.0±0.4 μM, and 1.7±0.3 μM respectively, indicating that glabrone has a more significant anti-inflammatory effect than glabridin.

[0067] Example 4

[0068] In this example, the inhibitory effect of glabrone on the activity of tyrosinase was tested.

[0069] Prepare 0.05 mol / L PBS (pH = 6.8), 10 mmol / L L-DOPA solution, 50 U / mL tyrosinase solution and different concentrations of glabrone in advance. Divide the experiment into four groups, namely the normal group, the normal zero-adjustment group, the experimental group, and the experimental zero-adjustment group. Among them, the specific treatment methods for each group are as follows:

[0070] Normal group: Precisely measure 50 μL of tyrosinase solution and 50 μL of distilled water into a 96-well plate. After mixing, incubate at 28 °C in a water bath for 15 min, then add 100 μL of L-DOPA, react at room temperature for 10 min, and measure the absorbance at 475 nm. The obtained absorbance is recorded as A1;

[0071] Normal zero-adjustment group: Precisely measure 100 μL of distilled water into a 96-well plate. After mixing, incubate at 28 °C in a water bath for 15 min, then add 100 μL of L-DOPA, react at room temperature for 10 min, and measure the absorbance at 475 nm. The obtained absorbance is recorded as A2;

[0072] Experimental group: Precisely measure 50 μL of tyrosinase solution and 50 μL of glabrone with different concentrations into a 96-well plate. After mixing, incubate at 28 °C in a water bath for 15 min, then add 100 μL of L-DOPA, react at room temperature for 10 min, and measure the absorbance at 475 nm. The obtained absorbance is recorded as A3;

[0073] Experimental zero-adjustment group: Precisely measure 50 μL of distilled water and 50 μL of glabrone with different concentrations into a 96-well plate. After mixing, incubate at 28 °C in a water bath for 15 min, then add 100 μL of L-DOPA, react at room temperature for 10 min, and measure the absorbance at 475 nm. The obtained absorbance is recorded as A4;

[0074] Among them, in this example, the concentrations of glabrone used are 25, 50, and 100 μg / mL respectively.

[0075] At the same time, use glabridin with the same concentration range as a control for corresponding detection.

[0076] Use the following formula to calculate its tyrosinase inhibition rate:

[0077]

[0078] The detection results are as Figure 4 shown.

[0079] From Figure 4As can be seen from the results shown, under the condition of the same concentration, the inhibitory effect of glabrone on tyrosinase is weaker than that of glabridin (P<0.01). Further combining the influence on the amount of cellular melanin production in the above-mentioned examples and the analysis of the fact that enzyme expression levels often control through influencing signal pathways, it can be further speculated that the whitening effect of glabrone is not only achieved by inhibiting the activity of tyrosinase to reduce melanin production, but essentially also affects melanin production by regulating cellular signal pathways.

[0080] In summary, through the above experimental data, it can be shown that within the concentration range without cytotoxicity, glabrone has a more excellent inhibitory effect on cellular melanin production than glabridin, the main known whitening active ingredient in licorice. Moreover, the inhibitory effect of glabrone on melanin production is achieved through multiple pathways. At the same time, glabrone also has a good anti-inflammatory effect, and its anti-inflammatory effect is similar to that of glabridin, and even superior to glabridin in some cases. Moreover, in terms of toxicity, glabrone is safer than glabridin. Therefore, it can be seen that glabrone has good application prospects in the preparation of compositions for inhibiting skin melanin production and inflammation.

[0081] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of glabrone or its derivatives in the preparation of products for whitening, reducing melanin and / or anti-inflammatory purposes.

2. The use according to claim 1, wherein The derivatives include pharmaceutically acceptable salts, solvate compounds, tautomers or mixtures thereof.

3. The use according to claim 1, characterized in that, The products include pharmaceuticals and cosmetics.

4. The use according to claim 1, characterized in that, The products are topical products.

5. The use according to any one of claims 1-4, characterized in that, In the products, the content of glabrone or its derivatives is greater than or equal to 0.1 w / w%.

6. The use according to any one of claims 1 to 4, characterized in that, In the products, the content of glabrone or its derivatives is 0.1 - 20 w / w%.

7. The use according to any one of claims 1-4, characterized in that, The products further contain adjuvants, which include pharmaceutically or cosmetically acceptable adjuvants.

8. The use according to claim 7, characterized in that, The adjuvants include at least one of antioxidants, preservatives, bactericides, humectants, emulsifiers, fragrances or pigments.

9. The use according to any one of claims 1 to 4, characterized in that, The products further contain a second active ingredient, and the function of the second active ingredient is the same as or different from that of glabrone or its derivatives.

10. The use according to any one of claims 1-4, characterized in that, The whitening and reducing melanin include inhibiting melanin production and / or reducing tyrosinase activity.

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