Skin melanin inhibitors, methods of making and using same

Abstract

The application belongs to the technical field of skin melanin inhibitors, and particularly relates to a skin melanin inhibitor, a preparation method and application thereof, wherein the inhibitor is a pterostilbene-phosphatidylcholine deep eutectic solvent, the pterostilbene-phosphatidylcholine deep eutectic solvent comprises pterostilbene and phosphatidylcholine, the pterostilbene-phosphatidylcholine DES combines with a supramolecular microcapsule technology to further improve the anti-inflammatory effect of pterostilbene; the pterostilbene-phosphatidylcholine DES and the pterostilbene-phosphatidylcholine supramolecular microcapsule can both improve the inhibition of pterostilbene on skin tumors, and the effect of the supramolecular microcapsule is more significant; meanwhile, the pterostilbene-phosphatidylcholine DES has a higher anti-inflammatory rate, so that the use dosage can be reduced, the effect is increased with a reduced amount, and the material cost is saved.

Description

Technical Field

[0001] This invention belongs to the field of skin melanin inhibitor technology, specifically relating to a skin melanin inhibitor, its preparation method, and its application. Background Technology

[0002] Melanin is formed from tyrosine or 3,4-dihydroxyphenylalanine through a series of chemical reactions. Animals, plants, and protists all possess this same pigment. Melanin is actually an amino acid derivative, present in everyone's body. The active period for melanin deposition is between the ages of 20 and 25. It exists in the cells of the basal layer of the skin, and is a substance called "melanin precursor," also known as "melanocyte" (or melanocyte matrix).

[0003] Melanocytes (melanocyte matrix) are animal cells that contain melanin or other similar pigments. They are typically located in the epidermis of the skin and the uvea (the pigmented layer behind the iris) of the eye. The color of the skin, hair, and eyes, as well as blemishes, acne scars, and pigmentation on the skin such as moles, freckles, etc., are all related to melanocytes (melanocyte matrix).

[0004] Recent research indicates that melanin matrix in different types of spots is more active than melanin matrix in surrounding non-spot areas, characterized by abnormally large dendrites in the melanin matrix. Therefore, by stabilizing or inhibiting the activity of the melanin matrix, it is possible to suppress melanin synthesis, which can help with skin inflammation and tumor cell formation issues such as spots, acne scars, and pigmentation.

[0005] Effective ingredients that inhibit melanin formation include chloroquine, indomethacin, vitamin C, beta-carotene, green tea, kojic acid, melanin, dried plum, cinnamon, beta-glucan, licorice oil, and soluble extracts. However, there are currently few substances or drugs that can effectively inhibit skin melanin, and further research is needed on other substances that inhibit skin melanin. Summary of the Invention

[0006] To address the problems in the prior art, this invention provides a skin melanin inhibitor, its preparation method, and its application, achieving the goal of enhancing the inhibition rate of pterostilbene against COX-2 and promoting the inhibitory effect of pterostilbene on skin tumors by preparing DES from pterostilbene and phosphatidylcholine.

[0007] The technical problem solved by this invention is achieved by the following technical solution:

[0008] The purpose of this invention is to provide a skin melanin inhibitor, wherein the inhibitor is a pterostilbene-phosphatidylcholine eutectic solvent, and the pterostilbene-phosphatidylcholine eutectic solvent includes pterostilbene and phosphatidylcholine.

[0009] Pterostilbene is an active ingredient derived from plants such as sandalwood, blueberry, grape, and rosewood. Pterostilbene has anticancer, anti-inflammatory, antioxidant, and analgesic effects. It can effectively inhibit the expression of the inflammatory factor cyclooxygenase-2 (COX-2), promote apoptosis of cancer cells, and inhibit skin tumors. Simultaneously, pterostilbene has the ability to inhibit NF-κB activation, thereby reducing the secretion of IL-1β, IL-6, and NO, and alleviating inflammatory stimulation. However, the effectiveness of using pterostilbene to inhibit skin melanin production still needs improvement.

[0010] Eutectic solvents (DES) consist of two main components: hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs). HBDs are such as acids, amines, or alcohols, while HBAs are such as tetraalkylammonium salts, quaternary ammonium salts, or phosphonium salts. Forming eutectic solvents with pterostilbene and phosphatidylcholine further enhances the effect of inhibiting melanin production in the skin.

[0011] Furthermore, the mass percentages of pterostilbene and phosphatidylcholine were 10%:90% (w / w) to 90%:10% (w / w).

[0012] Furthermore, the inhibitor is pterostilbene-phosphatidylcholine supramolecular microcapsules, which include a pterostilbene-phosphatidylcholine eutectic solvent.

[0013] Supramolecular microencapsulation technology is built upon liposomes, microcapsules, ionic liquid stabilization technology, dermal cell-targeted release technology, hair follicle-targeted release technology, and inflammatory factor-responsive release technology. The product precisely delivers active ingredients by creating artificial transport channels. It exhibits excellent transdermal absorption, long retention time at the skin target site, and good stability. A supramolecular pterostilbene microcapsule was prepared using the above-mentioned pterostilbene-phosphatidylcholine DES, further effectively promoting the expression of the antioxidant and anti-inflammatory effects of pterostilbene.

[0014] Furthermore, the pterostilbene-phosphatidylcholine supramolecular microcapsules also include emulsifiers, humectants, and water.

[0015] Furthermore, the mass percentages of each component in the pterostilbene-phosphatidylcholine supramolecular microcapsules are as follows: pterostilbene-phosphatidylcholine DES: 0.1-30%, emulsifier: 0.1-10%, humectant: 1-40%, and pure water: balance.

[0016] Furthermore, the emulsifier is at least one of PEG-40 hydrogenated castor oil, Tween 80, PEG-100 glyceryl stearate, glyceryl stearate, stearyl alcohol polyether-21, sorbitan polyether-30 tetraoleate, cetearyl glucoside, cetearyl alcohol, polyglycerol-10 laurate, and behenol; and the humectant is at least one of glycerin, butylene glycol, propylene glycol, 1,3-propanediol, dipropylene glycol, ethoxydiethylene glycol, xylitol, betaine, pentylene glycol, and hexanediol.

[0017] DES can be prepared by heating a mixture of HBA and HBD until a homogeneous, transparent solution is formed. As described below, the melting point of DES is significantly lower than that of its individual components.

[0018] A method for preparing a eutectic solvent for a skin melanin inhibitor, pterostilbene-phosphatidylcholine, includes the following steps:

[0019] (1) Clean the reactor, dry it, turn on the stirring and heating, and heat it at 90-110℃. Inert gas is introduced for protection.

[0020] (2) Add pterostilbene and phosphatidylcholine in proportion, and after melting, keep stirring and inert gas protection, and continue to keep warm for 6-8 hours to obtain pterostilbene-phosphatidylcholine eutectic solvent.

[0021] DES can also be prepared by dissolving the individual components in a solvent and then evaporating the solvent. As described below, the melting point of DES is significantly lower than that of its individual components.

[0022] A method for preparing a eutectic solvent for a skin melanin inhibitor, pterostilbene-phosphatidylcholine, includes the following steps:

[0023] (1) Clean the reactor, dry it, turn on the stirring and heating, and heat it at 50-60℃. Inert gas is introduced for protection.

[0024] (2) Add pterostilbene and phosphatidylcholine in proportion, add ethanol, and after melting, keep stirring and inert gas protection, and continue to keep warm for 1-2 hours to obtain a eutectic solvent.

[0025] (3) Ethanol was removed by rotary evaporation at 50℃ to obtain the eutectic solvent of pterostilbene-phosphatidylcholine.

[0026] A method for preparing supramolecular microcapsules of pterostilbene-phosphatidylcholine, a skin melanin inhibitor, includes the following steps:

[0027] (1) Preparation of oil phase: Weigh out the eutectic solvent of hydroxypsylarin-phosphatidylcholine and emulsifier, heat to 60-80℃, stir to dissolve and mix thoroughly to obtain the oil phase;

[0028] (2) Preparation of aqueous phase: Weigh the humectant and water, heat to 60-80℃, stir to dissolve and mix thoroughly to obtain the aqueous phase;

[0029] (3) Preparation of primary emulsion by shearing: The oil phase obtained above is added to the aqueous phase and sheared for 2-4 min at a speed of 5000-12000 rpm to obtain pterostilbene-phosphatidylcholine supramolecular microcapsule primary emulsion.

[0030] (4) Homogenized pterostilbene-phosphatidylcholine supramolecular microcapsules: The colostrum obtained above was homogenized at 400-800 bar, cyclicated 5-8 times, and cooled to room temperature to obtain pterostilbene-phosphatidylcholine supramolecular microcapsules.

[0031] The application of a skin melanin inhibitor in the preparation of skin melanin-inhibiting drugs or in the preparation of cosmetics. The pterostilbene-phosphatidylcholine DES and pterostilbene-phosphatidylcholine supramolecular microcapsules have the advantages of low cost and high efficacy in the fields of cosmetics, functional foods and pharmaceuticals.

[0032] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0033] The pterostilbene-phosphatidylcholine DES of this invention can effectively promote the anti-inflammatory effects of pterostilbene; the combination of pterostilbene-phosphatidylcholine DES with supramolecular microcapsule technology further enhances the anti-inflammatory effects of pterostilbene; both pterostilbene-phosphatidylcholine DES and pterostilbene-phosphatidylcholine supramolecular microcapsules can enhance the inhibition of skin melanin by pterostilbene, and the supramolecular microcapsules have a more significant effect; at the same time, because pterostilbene-phosphatidylcholine DES has a more efficient anti-inflammatory rate, the dosage can be reduced, reducing the amount used while increasing the effect, and saving material costs.

[0034] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and in order to make the above contents, objectives, features and advantages of the present invention more obvious and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0035] Figure 1 This is a diagram of the eutectic solvent of pterostilbene-phosphatidylcholine in this invention.

[0036] Figure 2 This is a diagram of the supramolecular microcapsules of pterostilbene-phosphatidylcholine in this invention.

[0037] Figure 3 DSC spectra of pterostilbene monomer, phosphatidylcholine monomer, and pterostilbene-phosphatidylcholine DES.

[0038] Figure 4The 1H-NMR spectra of pterostilbene monomer, phosphatidylcholine monomer, and pterostilbene-phosphatidylcholine DES are shown.

[0039] Figure 5 This is an electron micrograph of the pterostilbene-phosphatidylcholine supramolecular microcapsules of the present invention.

[0040] Figure 6 This is an image showing the appearance and stability of the pterostilbene-phosphatidylcholine supramolecular microcapsules in this invention.

[0041] Figure 7 This is a graph showing the particle size stability of the pterostilbene-phosphatidylcholine supramolecular microcapsules in this invention.

[0042] Figure 8 The graph shows the expression results of IL-6, IL-1β, and β-actin proteins in each experimental group during immunoblotting.

[0043] Figure 9 This is a graph showing the expression results of COX-2 protein in each experimental group during Western blot analysis. Detailed Implementation

[0044] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0045] In addition, unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or prepared by existing methods.

[0046] Example 1: Preparation of Pterostilbene-phosphatidylcholine DES

[0047] (1) Clean the reactor, dry it, turn on the stirring and heating, heat the temperature to 110°C, and introduce inert gas for protection;

[0048] (2) Add pterostilbene: phosphatidylcholine (10%: 90% w / w), and after melting, keep stirring and inert gas protection, and continue to keep warm for 8 hours to obtain pterostilbene-phosphatidylcholine DES.

[0049] Example 2: Preparation of Pterostilbene-phosphatidylcholine DES

[0050] (1) Clean the reactor, dry it, turn on the stirring and heating, heat the temperature to 100°C, and introduce inert gas for protection;

[0051] (2) Add pterostilbene: phosphatidylcholine (20%: 80% w / w), and after melting, keep stirring and inert gas protection, and continue to keep warm for 7 hours to obtain pterostilbene-phosphatidylcholine DES.

[0052] Example 3: Preparation of Pterostilbene-phosphatidylcholine DES

[0053] (1) Clean the reactor, dry it, turn on the stirring and heating, heat the temperature to 90°C, and introduce inert gas for protection;

[0054] (2) Add pterostilbene: phosphatidylcholine (33.3%: 66.7% w / w), and after melting, keep stirring and inert gas protection, and continue to keep warm for 6 hours to obtain pterostilbene-phosphatidylcholine DES.

[0055] Example 4: Preparation of Pterostilbene-phosphatidylcholine DES

[0056] (1) Clean the reactor, dry it, turn on the stirring and heating, heat the temperature to 100°C, and introduce inert gas for protection;

[0057] (2) Add pterostilbene:phosphatidylcholine (50%:50% w / w), and after melting, keep stirring and inert gas protection, and continue to keep warm for 7 hours to obtain pterostilbene-phosphatidylcholine DES.

[0058] Example 5: Preparation of Pterostilbene-phosphatidylcholine DES

[0059] (1) Clean the reactor, dry it, turn on the stirring and heating, heat the temperature to 100°C, and introduce inert gas for protection;

[0060] (2) Add pterostilbene: phosphatidylcholine (80%: 20% w / w), and after melting, keep stirring and inert gas protection, and continue to keep warm for 7 hours to obtain pterostilbene-phosphatidylcholine DES.

[0061] Example 6: Preparation of Pterostilbene-phosphatidylcholine DES

[0062] (1) Clean the reactor, dry it, turn on the stirring and heating, heat the temperature to 90°C, and introduce inert gas for protection;

[0063] (2) Add pterostilbene:phosphatidylcholine (90%:10% w / w), and after melting, keep stirring and inert gas protection, and continue to keep warm for 7 hours to obtain pterostilbene-phosphatidylcholine DES.

[0064] Example 7: Preparation of Pterostilbene-phosphatidylcholine DES

[0065] (1) Clean the reactor, dry it, turn on the stirring and heating, and heat it at 50°C. Inert gas is introduced for protection.

[0066] (2) Pterostilbene:phosphatidylcholine (10%:90% w / w), a certain amount of ethanol was added, and after melting, it was stirred and protected with inert gas for 1 hour to obtain DES.

[0067] (3) Ethanol was removed by rotary evaporation at 50℃ to obtain pterostilbene-phosphatidylcholine DES.

[0068] Example 8: Preparation of Pterostilbene-phosphatidylcholine DES

[0069] (1) Clean the reactor, dry it, turn on the stirring and heating, and heat it to 60°C. Inert gas is introduced for protection.

[0070] (2) Pterostilbene:phosphatidylcholine (20%:80% w / w), a certain amount of ethanol was added, and after melting, it was stirred and protected with inert gas for 2 hours to obtain DES.

[0071] (3) Ethanol was removed by rotary evaporation at 50℃ to obtain pterostilbene-phosphatidylcholine DES.

[0072] Example 9: Preparation of Pterostilbene-phosphatidylcholine DES

[0073] (1) Clean the reactor, dry it, turn on the stirring and heating, and heat it at 50°C. Inert gas is introduced for protection.

[0074] (2) Pterostilbene:phosphatidylcholine (33.3%:66.6% w / w), a certain amount of ethanol was added, and after melting, the mixture was stirred and kept under inert gas protection for 1.5 hours to obtain DES;

[0075] (3) Ethanol was removed by rotary evaporation at 50℃ to obtain pterostilbene-phosphatidylcholine DES.

[0076] Example 10: Preparation of Pterostilbene-phosphatidylcholine DES

[0077] (1) Clean the reactor, dry it, turn on the stirring and heating, and heat it at 50°C. Inert gas is introduced for protection.

[0078] (2) Pterostilbene:phosphatidylcholine (50%:50% w / w), add a certain amount of ethanol, and after melting, keep stirring and inert gas protection, continue to keep warm for 2 hours to obtain DES;

[0079] (3) Ethanol was removed by rotary evaporation at 50℃ to obtain pterostilbene-phosphatidylcholine DES.

[0080] Example 11: Preparation of Pterostilbene-phosphatidylcholine DES

[0081] (1) Clean the reactor, dry it, turn on the stirring and heating, and heat it at 50°C. Inert gas is introduced for protection.

[0082] (2) Pterostilbene:phosphatidylcholine (80%:20% w / w), a certain amount of ethanol was added, and after melting, the mixture was stirred and protected with inert gas for 1 hour to obtain DES.

[0083] (3) Ethanol was removed by rotary evaporation at 50℃ to obtain pterostilbene-phosphatidylcholine DES.

[0084] Example 12: Preparation of Pterostilbene-phosphatidylcholine DES

[0085] (1) Clean the reactor, dry it, turn on the stirring and heating, and heat it at 50°C. Inert gas is introduced for protection.

[0086] (2) Pterostilbene:phosphatidylcholine (90%:10% w / w), a certain amount of ethanol was added, and after melting, the mixture was stirred and protected with inert gas for 1 hour to obtain DES.

[0087] (3) Ethanol was removed by rotary evaporation at 50℃ to obtain pterostilbene-phosphatidylcholine DES.

[0088] Example 13: Preparation of a supramolecular microcapsule of pterostilbene-phosphatidylcholine

[0089] (1) Preparation of the oil phase:

[0090] Weigh out 0.1% of pterostilbene-phosphatidylcholine (33.3%: 66.7% w / w) DES and 0.1% of cetearyl glucoside as emulsifier. Heat to the set temperature of 60°C and stir to dissolve and mix thoroughly to obtain the oil phase.

[0091] (2) Preparation of aqueous phase:

[0092] Weigh out the following humectant: 1% butylene glycol and 98.8% water. Heat to the set temperature of 60°C and stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0093] (3) High-speed shearing preparation of primary emulsion: The oil phase obtained above was added to the aqueous phase and sheared at high speed for 2 min at a speed of 5000 rpm to obtain the pterostilbene-phosphatidylcholine supramolecular microcapsule primary emulsion.

[0094] (4) High-pressure homogenization to prepare pterostilbene-phosphatidylcholine supramolecular microcapsules: The colostrum obtained above was homogenized at 400 bar for 5 cycles and cooled to room temperature to obtain pterostilbene-phosphatidylcholine supramolecular microcapsules.

[0095] Example 14: Preparation of a supramolecular microcapsule of pterostilbene-phosphatidylcholine

[0096] (1) Preparation of the oil phase:

[0097] Weigh out 3% of pterostilbene-phosphatidylcholine (33.3%:66.7% w / w), 1% of cetearyl glucoside, and heat to the set temperature of 60°C. Stir to dissolve and mix thoroughly to obtain the oil phase.

[0098] (2) Preparation of aqueous phase:

[0099] Weigh out the following humectant: 5% butylene glycol and 91% water. Heat to the set temperature of 60°C and stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0100] (3) High-speed shearing preparation of primary emulsion: The oil phase obtained above was added to the aqueous phase and sheared at high speed for 2 min at a speed of 5000 rpm to obtain the pterostilbene-phosphatidylcholine supramolecular microcapsule primary emulsion.

[0101] (4) High-pressure homogenization to prepare pterostilbene-phosphatidylcholine supramolecular microcapsules: The colostrum obtained above was homogenized at 400 bar for 5 cycles and cooled to room temperature to obtain pterostilbene-phosphatidylcholine supramolecular microcapsules.

[0102] Example 15: Preparation of a supramolecular microcapsule of pterostilbene-phosphatidylcholine

[0103] Weigh out 12% of pterostilbene-phosphatidylcholine (33.3%:66.7% w / w), DES, 2% of cetearyl glucoside, and 1% of PEG-40 hydrogenated sesame oil. Heat to a set temperature of 60°C and stir to dissolve and mix thoroughly to obtain the oil phase.

[0104] (2) Preparation of aqueous phase:

[0105] Weigh out the following moisturizers: butylene glycol: 5%, glycerin: 5%, and water: 75%. Heat to the set temperature of 70°C and stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0106] (3) High-speed shearing preparation of primary emulsion: The oil phase obtained above was added to the aqueous phase and sheared at high speed for 3 min at a speed of 10000 rpm to obtain the pterostilbene-phosphatidylcholine supramolecular microcapsule primary emulsion.

[0107] (4) High pressure homogenization to prepare pterostilbene-phosphatidylcholine supramolecular microcapsules: The colostrum obtained above was homogenized at 600 bar for 7 cycles and cooled to room temperature to obtain pterostilbene-phosphatidylcholine supramolecular microcapsules.

[0108] Example 16: Preparation of a supramolecular microcapsule of pterostilbene-phosphatidylcholine

[0109] (1) Preparation of the oil phase:

[0110] Weigh out 15% of pterostilbene-phosphatidylcholine (33.3%:66.7% w / w), 2% of PEG-40 hydrogenated sesame oil, and 3% of polyglycerol-10 laurate. Heat to a set temperature of 60°C and stir to dissolve and mix thoroughly to obtain the oil phase.

[0111] (2) Preparation of aqueous phase:

[0112] Weigh out the following humectants: butylene glycol: 5%, glycerin: 5%, propylene glycol: 10%, and water: 60%. Heat to the set temperature of 80°C and stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0113] (3) High-speed shearing preparation of primary emulsion: The oil phase obtained above was added to the aqueous phase and sheared at high speed for 4 min at a speed of 12000 rpm to obtain the pterostilbene-phosphatidylcholine supramolecular microcapsule primary emulsion.

[0114] (4) High pressure homogenization to prepare pterostilbene-phosphatidylcholine supramolecular microcapsules: The colostrum obtained above was homogenized at 800 bar for 8 cycles and cooled to room temperature to obtain pterostilbene-phosphatidylcholine supramolecular microcapsules.

[0115] Example 17: Preparation of a supramolecular microcapsule of pterostilbene-phosphatidylcholine

[0116] (1) Preparation of the oil phase:

[0117] Weigh out pterostilbene-phosphatidylcholine (33.3%: 66.7% w / w), DES: 30%.

[0118] Emulsifiers: PEG-40 hydrogenated sesame oil: 2%, polyglycerol-10 laurate: 3%, sorbitol polyether-30 tetraoleate: 3%, behenol: 2%;

[0119] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the oil phase.

[0120] (2) Preparation of aqueous phase:

[0121] Weigh out the following moisturizers: butylene glycol: 10%, glycerin: 5%, propylene glycol: 10%, pentanediol: 5%, dipropylene glycol: 10%, and water: 20%. Heat to the set temperature of 80°C and stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0122] (3) High-speed shearing preparation of primary emulsion: The oil phase obtained above was added to the aqueous phase and sheared at high speed for 4 min at a speed of 12000 rpm to obtain the pterostilbene-phosphatidylcholine supramolecular microcapsule primary emulsion.

[0123] (4) High pressure homogenization to prepare pterostilbene-phosphatidylcholine supramolecular microcapsules: The colostrum obtained above was homogenized at 800 bar for 8 cycles and cooled to room temperature to obtain pterostilbene-phosphatidylcholine supramolecular microcapsules.

[0124] Experimental Example 1: Preparation of Pterostilbene Solution

[0125] Weigh out 4% Pterostilbene and 20% DMOS, stir until dissolved; add 76% water to obtain the final product.

[0126] Experimental Example 2: Preparation of Pterostilbene-phosphatidylcholine DES solution

[0127] Weigh out 12% of pterostilbene-phosphatidylcholine DES and 20% of DMOS from Example 3, stir until dissolved, and add 68% water to obtain the final product.

[0128] Test Methods Section

[0129] 1. Determination of melting point using DSC differential scanning calorimetry

[0130] The pterostilbene-phosphatidylcholine DES, pterostilbene monomer, and phosphatidylcholine monomer obtained in Example 4 were subjected to a temperature range of -50°C to 200°C, a heating rate of 10°C / min, and nitrogen protection to obtain their DSC spectra.

[0131] The results are as follows Figure 3 As shown, the results indicate that the prepared DES solvent containing pterostilbene-phosphatidylcholine is homogeneous and has a significantly reduced melting point.

[0132] 2. Detection by proton nuclear magnetic resonance (NMR) spectroscopy

[0133] Nuclear magnetic resonance spectrometer: Bruker, Switzerland, AVANCE NEO 400;

[0134] Frequency: 400MHz;

[0135] Injection volume: 5 mg;

[0136] Solvent: Deuterated DMSO.

[0137] Each of the following 5 mg of pterostilbene monomer, phosphatidylcholine monomer, and the pterostilbene-phosphatidylcholine DES obtained in Example 4 was dissolved in 0.6 mL of deuterated DMSO reagent and detected by 1H NMR spectroscopy.

[0138] The result is as follows Figure 4 As shown, the results indicate that the hydrogen peak position of the eutectic solvent is shifted compared to that of each monomer, proving that the eutectic solvent is a new eutectic substance rather than a physical mixture.

[0139] 3. Observation using transmission electron microscopy

[0140] The pterostilbene-phosphatidylcholine supramolecular microcapsules obtained in Example 15 were diluted with distilled water, and the sample was aspirated by capillary tube and blown onto a support grid. After staining, washing, and drying, the samples were observed using a transmission electron microscope, and the results were photographed and recorded. The results are as follows: Figure 5 As shown.

[0141] As shown in the figure, the prepared pterostilbene-phosphatidylcholine supramolecular microcapsule nanoparticles are uniformly dispersed, round or elliptical in shape, and have a particle size of about 120 nm.

[0142] 4. Stability detection of pterostilbene-phosphatidylcholine supramolecular microcapsules

[0143] Light stability chamber: STIK drug stability test chamber IMT-250L;

[0144] Thermal stability chamber: Boxun Pharmaceutical Stability Test Chamber BXY-2501;

[0145] 5℃ Refrigerated Box: Haier Laboratory Explosion-proof Refrigerated Box HLR-310FL;

[0146] -13℃ Freezer: Midea BD / BC-203KMD(E);

[0147] Nanoparticle size zeta potential analyzer: Anton Paar.

[0148] Experimental Method: Example 15 was dispensed into 15ml transparent PE bottles and placed in a light chamber at 45℃, 5℃, -13℃, 25℃, and after 7, 14, 28, and 60 days, the appearance was observed and the particle size was measured. Specific results are as follows: Figure 6 , 7 .

[0149] The results showed that the pterostilbene-phosphatidylcholine supramolecular microcapsules had good stability under various conditions.

[0150] 5. Immunoblotting detection

[0151] 5.1 Test Methods:

[0152] (1) After culturing the cells in a 37℃, 5% CO2 incubator for 48h, the cell pellet was collected and RIPA lysis buffer was added for lysis at 4℃ for 30min. The supernatant was collected by centrifugation at 12000rpm / 10min and the total cell protein was extracted.

[0153] (2) Protein concentration was determined by the BCA method;

[0154] (3) Take 40 μg of protein sample and mix it with 5x loading buffer (volume ratio 4:1), and then perform SDS-PAGE electrophoresis to separate the protein.

[0155] (4) Protein electrotransfer onto PVDF membrane, sealed with 5% skim milk powder for 1 hour;

[0156] (5) After adding the primary antibody and incubating overnight at 4°C, rinse with TBST 3 times, 10 min each time;

[0157] (6) The next day, add horseradish peroxidase-labeled secondary antibody [Goat Anti-Rabbit IgG(H+L)HRP(1:5000)], incubate at 37°C for 1 hour, and wash 3 times with TBST for 10 minutes each time;

[0158] (7) The protein was detected by ECL substrate chemiluminescence imaging. The results were quantitatively analyzed using Quantity One software. The expression level of the protein was expressed by the ratio of the integral absorbance.

[0159] β-actin was used as an internal control. For target protein quantification, band intensity was converted to optical density, normalized using β-actin values, and the protein level of β-actin was defined as 100%.

[0160] 5.2 Human Epidermal Cell Inflammatory Factor Inhibition Experiment

[0161] Following the experimental method in 5.1, human epidermal cells were selected and primary antibodies IL-1β (1:1000) and IL-6 (1:1000) were added to obtain the negative control group (NC).

[0162] Positive control group (AC): During the cell culture process in (1), the positive control group was obtained by UV (UVA51, UVB49) irradiation according to the above method.

[0163] Experimental Group 1 (DES): Following the above method, during cell culture (1), UV (UVA51, UVB49) light was applied, and 10 μL of the pterostilbene-phosphatidylcholine DES solution from Experimental Example 2 was added.

[0164] Experimental group 2 (MC): Following the above method, during the cell culture process (1), UV (UVA51, UVB49) light was applied, and 10 μL of the pterostilbene-phosphatidylcholine supramolecular microcapsules obtained in Example 15 were added.

[0165] Experimental group 3 (PTS): Following the above method, during the cell culture process in (1), UV (UVA51, UVB49) light was applied, and 1 μL of the pterostilbene solution from Experimental Example 1 was added.

[0166] 5.3 COX-2 detection in melanoma cells

[0167] Following the method in 5.1, melanoma cells were selected and COX-2 (1:1000) was added as the primary antibody to obtain the negative control group (NC).

[0168] Experimental group 1 (DES): Following the above method, 10 μL of the pterostilbene-phosphatidylcholine DES solution from Experiment Example 2 was added during cell culture in (1).

[0169] Experimental group 2 (MC): Following the above method, 10 μL of the pterostilbene-phosphatidylcholine supramolecular microcapsules obtained in Example 15 were added during cell culture in (1).

[0170] Experimental group 3 (PTS): Following the above method, 10 μL of the pterostilbene solution from Experimental Example 1 was added during cell culture in (1).

[0171] The NC group was used as an internal control. For target protein quantification, band intensity was converted to optical density, normalized using values ​​from the NC group, and the protein level in the NC group was defined as 100%.

[0172] The above experiment was conducted in 6 parallel groups, and the resulting bar graphs were obtained. Figure 8 , Figure 9 The results summarized from the stripes are shown in Table 1 below:

[0173] Table 1

[0174]

[0175] Note: ± represents the standard deviation.

[0176] The results show that:

[0177] (1) Pterostilbene-phosphatidylcholine DES can effectively inhibit the expression of inflammatory factors IL-6 and IL-1β in human epidermal cells and enhance the anti-inflammatory effect of pterostilbene.

[0178] (2) Pterostilbene-phosphatidylcholine supramolecular microcapsules can further inhibit the expression of IL-6 and IL-1β in human epidermal cells, thus enhancing the anti-inflammatory effect of pterostilbene.

[0179] (3) Pterostilbene-phosphatidylcholine DES can enhance the inhibitory effect of pterostilbene on melanoma cells.

[0180] (4) Pterostilbene-phosphatidylcholine supramolecular microcapsules can further enhance the inhibitory effect of pterostilbene on melanoma cells.

[0181] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0182] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A skin melanin inhibitor, characterized in that, The inhibitor is a pterostilbene-phosphatidylcholine eutectic solvent, which includes pterostilbene and phosphatidylcholine. The mass percentages of pterostilbene and phosphatidylcholine were 10%:90% (w / w) to 90%:10% (w / w); The pterostilbene-phosphatidylcholine eutectic solvent was prepared by heating and melting a mixture of pterostilbene and phosphatidylcholine; Alternatively, the eutectic solvent of pterostilbene and phosphatidylcholine can be prepared by dissolving pterostilbene and phosphatidylcholine in a solvent, heating to melt, and then evaporating the solvent.

2. The skin melanin inhibitor as described in claim 1, characterized in that: The inhibitor is pterostilbene-phosphatidylcholine supramolecular microcapsules, which contain a pterostilbene-phosphatidylcholine eutectic solvent.

3. The skin melanin inhibitor as described in claim 2, characterized in that: The pterostilbene-phosphatidylcholine supramolecular microcapsules also include emulsifiers, humectants, and water.

4. The skin melanin inhibitor as described in claim 3, characterized in that: The mass percentages of each component in the pterostilbene-phosphatidylcholine supramolecular microcapsules are as follows: Pterostilbene-phosphatidylcholine DES: 0.1-30%, emulsifier: 0.1-10%, humectant: 1-40%, and pure water: balance.

5. The skin melanin inhibitor as described in claim 4, characterized in that: The emulsifier is at least one of PEG-40 hydrogenated castor oil, Tween 80, PEG-100 glyceryl stearate, glyceryl stearate, stearyl alcohol polyether-21, sorbitan polyether-30 tetraoleate, cetearyl glucoside, cetearyl alcohol, polyglycerol-10 laurate, and behenol. The humectant is at least one of glycerin, butylene glycol, propylene glycol, 1,3-propanediol, dipropylene glycol, ethoxydiethylene glycol, xylitol, betaine, pentylene glycol, and hexanediol.

6. The method for preparing a skin melanin inhibitor as described in claim 1, characterized in that: The preparation method of the pterostilbene-phosphatidylcholine eutectic solvent includes the following steps: (1) Clean the reactor, dry it, turn on the stirring and heating, and heat it at 90-110℃. Inert gas is introduced for protection. (2) Add pterostilbene and phosphatidylcholine in proportion, and after melting, keep stirring and inert gas protection, and continue to keep warm for 6-8 hours to obtain pterostilbene-phosphatidylcholine eutectic solvent.

7. The method for preparing a skin melanin inhibitor as described in claim 1, characterized in that: The preparation method of the pterostilbene-phosphatidylcholine eutectic solvent includes the following steps: (1) Clean the reactor, dry it, turn on the stirring and heating, and heat it at 50-60℃. Inert gas is introduced for protection. (2) Add pterostilbene and phosphatidylcholine in proportion, add ethanol, and after melting, keep stirring and inert gas protection, and continue to keep warm for 1-2 hours to obtain a eutectic solvent; (3) Ethanol was removed by rotary evaporation at 50°C to obtain the eutectic solvent of pterostilbene-phosphatidylcholine.

8. The method for preparing a skin melanin inhibitor according to any one of claims 2-5, characterized in that: The preparation method of pterostilbene-phosphatidylcholine supramolecular microcapsules includes the following steps: (1) Preparation of oil phase: Weigh out the eutectic solvent of hydroxypsylarithrosine-phosphatidylcholine and emulsifier, heat to 60-80℃, stir to dissolve and mix thoroughly to obtain the oil phase; (2) Preparation of aqueous phase: Weigh the humectant and water, heat to 60-80℃, stir to dissolve and mix thoroughly to obtain the aqueous phase; (3) Shearing preparation: The oil phase obtained above is added to the aqueous phase and sheared for 2-4 min at a speed of 5000-12000 rpm to obtain the pterostilbene-phosphatidylcholine supramolecular microcapsule colostrum. (4) Homogenization: The colostrum obtained above is homogenized at 400-800 bar, 5-8 times, and cooled to room temperature to obtain pterostilbene-phosphatidylcholine supramolecular microcapsules.

9. The use of a skin melanin inhibitor as described in any one of claims 1-8 in the preparation of a skin melanin-inhibiting drug or in the preparation of a cosmetic.

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