Composition, preparation and application of 4-butylresorcinol nanoliposome

Abstract

The invention discloses a composition, preparation and application of 4-butylresorcinol nanoliposomes, and relates to the field of cosmetics. By selectively studying active substances, antioxidants, carriers and adjuvants, the active substances, antioxidants, carriers and adjuvants are mixed and homogenized and subjected to high-pressure microfluidization to obtain 4-butylresorcinol nanoliposomes. The active substance is 4-butylresorcinol, the carrier includes phospholipids, cholesterol and DSPE-PEG2000-LPTATH, and the adjuvants are caprylic / capric triglycerides, glycerol and water. The 4-butylresorcinol nanoliposomes prepared by the present invention have small particle size and PDI, high encapsulation efficiency and targeting, and have good inhibitory effect on the relative activity of tyrosinase and the generation of melanin, and have high stability.

Description

Technical Field

[0001] The invention relates to the field of cosmetics, and in particular to the composition, preparation and application of 4-butylresorcinol nanoliposomes. Background Art

[0002] The differences in skin color among human bodies are mainly affected by genetic genes, which determine the type, quantity and distribution of melanin in the skin. Melanin is produced by epidermal melanocytes, which convert tyrosine into melanin through a series of complex biochemical reactions. This process is mainly catalyzed by three key enzymes: tyrosinase, tyrosinase-associated protein 1 and tyrosinase-associated protein 2. Tyrosine generates dopa under the catalysis of tyrosinase, and dopa further generates dopaquinone under the action of tyrosinase. After dopaquinone cyclizes itself, it generates melanin under the action of TRP-1 and TRP-2. Melanin is transferred along the extension direction of melanocyte dendrites and gradually transferred to the surrounding keratinocytes, causing changes in skin color.

[0003] 4-Butylresorcinol is a resorcinol derivative that inhibits the action of tyrosinase by competing for the active site of tyrosinase. It can also inhibit TRP-1 activity, reduce the production of melanin in the body, and achieve the purpose of whitening and lightening spots. 4-Butylresorcinol is more than 6 times stronger than phenylethylresorcinol in inhibiting tyrosinase activity and 310 times stronger than arbutin. However, 4-Butylresorcinol is irritating and can easily cause symptoms such as skin tingling, redness, and thinning of keratin. It also has poor transdermal absorption, is easily oxidized, easily discolored, and is unstable when exposed to light and heat. These have limited the application of 4-Butylresorcinol. Summary of the invention

[0004] The purpose of the present invention is to provide a composition, preparation and application of 4-butylresorcinol nanoliposomes to solve the problems of 4-butylresorcinol being easy to irritate the skin, easy to be oxidized and discolored and unstable.

[0005] In order to solve the above technical problems, the present invention specifically provides the following technical solutions:

[0006] The invention discloses a nanoliposome, comprising: an active substance, an antioxidant, a carrier and an auxiliary agent; the antioxidant comprises at least one of 3-o-ethyl ascorbic acid, ascorbyl palmitate, a vanillate derivative and hexadecyl citric acid. The use of the vanillate derivative and the hexadecyl citric acid can prevent the active substance in the nanoliposome and the nanoliposome itself from being oxidized, thereby improving the stability and protecting the active substance, prolonging the effectiveness of the nanoliposome, thereby better acting on tyrosinase and melanin, enhancing the inhibitory effect on tyrosinase and melanin, and delaying the chromaticity change of the nanoliposome itself.

[0007] Preferably, the active is 4-butylresorcinol.

[0008] Preferably, the carrier further comprises phospholipid, cholesterol and DSPE-PEG2000-LPTATH; the phospholipid is at least one of soybean lecithin, sunflower lecithin, hydrogenated lecithin, phosphatidylcholine and hydrogenated phosphatidylcholine; DSPE-PEG2000-LPTATH is obtained by covalently bonding DSPE-PEG2000-NHS and hexapeptide-2.

[0009] Preferably, the auxiliary agent comprises a lipid auxiliary agent and a water phase auxiliary agent, the lipid auxiliary agent is caprylic acid / capric acid triglyceride, and the water phase auxiliary agent is glycerin and water.

[0010] Preferably, the mass ratio of the active substance to the antioxidant is 1:0.1-0.5, the mass ratio of the active substance to the carrier is 1:0.3-1.8, and the mass ratio of the active substance to the auxiliary agent is 1:4.5-16.

[0011] The present invention discloses a method for preparing nanoliposomes, comprising:

[0012] Active substances, antioxidants, carriers and lipid additives are mixed to obtain phase A, and aqueous phase additives are mixed to obtain phase B; then phase A is added to phase B for homogenization, and then subjected to high-pressure microfluidization treatment to obtain nanoliposomes.

[0013] Preferably, the active substance is 4-butylresorcinol, the antioxidant is at least one of 3-o-ethylascorbic acid, ascorbyl palmitate, a vanillate derivative and hexadecyl citric acid, and the mass ratio of the amount of the active substance used to the amount of the antioxidant used is 1:0.1-0.5.

[0014] Preferably, the carrier comprises phospholipids, cholesterol and DSPE-PEG2000-LPTATH; the phospholipids are at least one of soybean lecithin, sunflower lecithin, hydrogenated lecithin, phosphatidylcholine and hydrogenated phosphatidylcholine; the lipid adjuvant is caprylic / capric triglyceride; and the aqueous phase adjuvant comprises glycerol and water.

[0015] Preferably, the mass ratio of the amount of active ingredient used to the amount of phospholipid used is 1:0.2-1.5.

[0016] Preferably, the mass ratio of the amount of active substance used to the amount of cholesterol used is 1:0.05-0.15.

[0017] Preferably, the mass ratio of the amount of the active substance used to the amount of DSPE-PEG2000-LPTATH used is 1:0.05-0.15.

[0018] Preferably, the mass ratio of the amount of the active ingredient used to the amount of the lipid auxiliary agent used is 1:1.5-2.

[0019] Preferably, the water phase adjuvant comprises glycerin and water.

[0020] More preferably, the mass ratio of the amount of active substance used to the amount of glycerol used is 1:1.2-3.5.

[0021] More preferably, the mass ratio of the amount of glycerol used to the amount of water used is 1:1.5-3.

[0022] Preferably, the homogenization speed of phase A and phase B is 2500-4000 r / min, the homogenization time is 1.5-5 min, and the pressure of the high-pressure microjet is 18000-30000 psi.

[0023] The invention discloses a cosmetic comprising the nano liposome or the nano liposome prepared by the method.

[0024] The present invention discloses a method for preparing nanoliposomes, comprising:

[0025] Active substances, antioxidants, carriers and lipid additives are mixed to obtain phase A, and aqueous phase additives are mixed to obtain phase B; then phase A is added to phase B for homogenization to obtain colostrum, and the colostrum is treated with high-pressure microfluidization to obtain nanoliposomes.

[0026] Preferably, in the above technical solution, the active substance is 4-butylresorcinol, and the antioxidant is at least one of 3-o-ethylascorbic acid, ascorbyl palmitate, a vanillate derivative and hexadecyl citric acid.

[0027] Preferably, in the above technical solution, the mass ratio of the amount of the active substance used to the amount of the antioxidant used is 1:0.1-0.5.

[0028] Preferably, in the above technical solution, the carrier comprises phospholipids, cholesterol and DSPE-PEG2000-LPTATH.

[0029] More preferably, in the above technical solution, the phospholipid is at least one of soybean lecithin, sunflower lecithin, hydrogenated lecithin, phosphatidylcholine and hydrogenated phosphatidylcholine.

[0030] Preferably, in the above technical solution, the mass ratio of the amount of active substance used to the amount of phospholipid used is 1:0.2-1.5.

[0031] Preferably, in the above technical solution, the mass ratio of the amount of active substance used to the amount of cholesterol used is 1:0.05-0.15.

[0032] Preferably, in the above technical solution, the lipid auxiliary agent is caprylic / capric triglyceride, and the mass ratio of the amount of the active ingredient used to the amount of the lipid auxiliary agent used is 1:1.5-2.

[0033] Preferably, in the above technical solution, the mass ratio of the amount of the active substance used to the amount of DSPE-PEG2000-LPTATH used is 1:0.05-0.15.

[0034] Preferably, in the above technical solution, the water phase auxiliary agent includes glycerol and water.

[0035] More preferably, the mass ratio of the amount of active substance used to the amount of glycerol used is 1:1.2-3.5.

[0036] More preferably, the mass ratio of the amount of glycerol used to the amount of water used is 1:1.5-3.

[0037] Preferably, in the above technical solution, the homogenization speed of phase A and phase B is 2500-4000 r / min, the homogenization time is 1.5-5 min, and the pressure of the high-pressure microjet is 18000-30000 psi.

[0038] Preferably, in the above technical solution, the high-pressure microfluidic treatment is performed 2-4 times.

[0039] Preferably, in the above technical solution, the colostrum is filtered at a micropore size of 0.2-0.3 μm after being treated with high-pressure microfluidization.

[0040] The present invention discloses a preparation method of DSPE-PEG2000-LPTATH, which is specifically:

[0041] First, DSPE-PEG2000-NHS and hexapeptide-2 were dissolved in a mixture of DMF and chloroform to obtain a reaction mixture, and then the pH was adjusted to 7.5-8.5, and the reaction was carried out under argon protection to obtain a reaction solution, the reaction solution was dialyzed to obtain a dialyzate, and the dialyzate was freeze-dried to obtain DSPE-PEG2000-LPTATH.

[0042] Preferably, the mass ratio of the amount of DSPE-PEG2000-NHS used to the amount of hexapeptide-2 used is 1:0.12-0.17.

[0043] Preferably, DMF and chloroform are mixed in a volume ratio of 1:0.8-1.2.

[0044] Preferably, the amount of DSPE-PEG2000-NHS in the mixed solution of DMF and chloroform is 0.087 g / ml.

[0045] Preferably, the solvent used for pH adjustment is N-methylmorpholine.

[0046] Preferably, the reaction temperature is 23-27° C., and the reaction time is 20-28 h.

[0047] Preferably, dialysis is performed using a 3500 MW dialysis bag.

[0048] Preferably, the dialysis time is 40-50 hours, and the deionized water is replaced every 4-6 hours.

[0049] The present invention discloses a method for preparing a vanillic acid ester derivative, which is specifically:

[0050] p-Methoxybenzylamine is added to a mixed solution of maleic anhydride and acetonitrile to react to obtain a crude product. Vanillate ester is mixed with dichloromethane to obtain a mixed solution, and the crude product, 2-methyl-1,3-propanediol, dicyclohexylcarbodiimide and 4-dimethylaminopyridine are added to the mixed solution to react to obtain a vanillate ester derivative.

[0051] Preferably, the mass ratio of the amount of p-methoxybenzylamine used to the amount of maleic anhydride used is 1:0.8-1.2.

[0052] Preferably, the amount of maleic anhydride in acetonitrile in the maleic anhydride acetonitrile mixed solution is 0.08-0.13 g / ml.

[0053] Preferably, the mass ratio of the amount of vanillic acid ester used to the amount of the crude product used is 1:0.7-1.5.

[0054] Preferably, the ratio of vanillic acid ester to 2-methyl-1,3-propanediol is 1:0.4-0.9.

[0055] Preferably, the mass ratio of the amount of vanillic acid ester to the amount of dicyclohexylcarbodiimide is 1:1-2.

[0056] Preferably, the mass ratio of the amount of vanillic acid ester to the amount of 4-dimethylaminopyridine is 1:0.02-0.1.

[0057] Preferably, in the process of preparing the crude product, after the reaction is completed, the mixture is cooled to 5-25° C. to precipitate solids, which are then filtered and dried to obtain the crude product.

[0058] Preferably, the reaction temperature of the mixed solution with the crude product, dicyclohexylcarbodiimide and 4-dimethylaminopyridine is 0-5°C, the reaction time is 5-8h, and after the reaction is completed, the mixture is filtered and evaporated to obtain the vanillic acid ester derivative.

[0059] More preferably, in the preparation of nanoliposomes, modified phospholipids can be added in addition to antioxidants. The antioxidants are vanillic acid ester derivatives and hexadecyl citric acid; the modified phospholipids can promote the release and cell uptake of active substances during the action process, thereby improving the effect of nanoliposomes, enhancing the inhibition of tyrosinase and melanin, and at the same time enhancing the structural stability of nanoliposomes, thereby reducing the chromaticity growth rate.

[0060] Preferably, the mass ratio of the amount of active substance used to the amount of modified phospholipid used is 1:0.2-1

[0061] The present invention also discloses a method for preparing the modified phospholipid, which is specifically:

[0062] Using n-hexane as solvent, phospholipid and conjugated linoleic acid ethyl ester react under the action of lipase to obtain modified phospholipid.

[0063] Preferably, the mass ratio of the amount of phospholipid used to the amount of conjugated linoleic acid ethyl ester used is 1:2.5-3.5.

[0064] Preferably, the mass ratio of the amount of phospholipid used to the amount of lipase used is 1:0.15-0.25.

[0065] Preferably, the reaction speed is 180-220 r / min, the reaction temperature is 50-57° C., and the reaction time is 20-30 h.

[0066] Preferably, after the reaction is completed, the reaction product needs to be centrifuged to separate the reaction solution, the centrifugal speed is 3500-5000r / min, and the centrifugal time is 8-15min.

[0067] Preferably, after the reaction is completed, the n-hexane needs to be evaporated and removed at an evaporation temperature of 48-55°C.

[0068] Preferably, after evaporation, cold acetone is added to wash 2-6 times to separate insoluble solids, and the insoluble solids are vacuum dried at 45-55° C. to obtain the modified phospholipids.

[0069] Compared with the prior art, the present invention has the following beneficial effects:

[0070] The 4-butylresorcinol nanoliposome prepared by the invention comprises an active substance, an antioxidant, a carrier and an auxiliary agent; the active substance is 4-butylresorcinol; the carrier comprises phospholipid, cholesterol and DSPE-PEG2000-LPTATH, and DSPE-PEG2000-LPTATH is obtained by covalently bonding DSPE-PEG2000-NHS and hexapeptide-2; the auxiliary agent is caprylic / capric triglyceride, glycerol and water; the active substance, the antioxidant, the carrier and the auxiliary agent are mixed and homogenized and subjected to high-pressure microfluidization treatment to obtain the 4-butylresorcinol nanoliposome. The 4-butylresorcinol nanoliposome prepared by the invention has a small particle size and PDI, a high encapsulation rate and targeting, and has a good inhibitory effect on the relative activity of tyrosinase and the generation of melanin, and has high stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0072] Figure 1 The figure is the result of the effect on cAMP content;

[0073] Figure 2 is the relative activity graph of tyrosinase;

[0074] Figure 3 This is a graph showing the relative content of melanin. DETAILED DESCRIPTION

[0075] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0076] The following first describes the concepts involved in the present application in conjunction with the accompanying drawings. It should be noted that the following description of each concept is only to make the content of the present application easier to understand, and does not limit the scope of protection of the present application; at the same time, the embodiments and features in the embodiments of the present application can be combined with each other in the absence of conflict. The present application will be described in detail with reference to the accompanying drawings and in conjunction with the embodiments.

[0077] Embodiment 1:

[0078] Preparation of 4-butylresorcinol nanoliposomes: 4-butylresorcinol, 3-o-ethyl ascorbic acid, ascorbyl tetraisopalmitate, hydrogenated lecithin, cholesterol and caprylic / capric triglyceride were stirred and mixed to obtain phase A. Glycerol and water were stirred and mixed to obtain phase B. Then phase A was added to phase B and homogenized at 3000 r / min for 2 min to obtain colostrum. Then, the colostrum was subjected to high-pressure microfluidization at 20000 psi. After repeated homogenization by high-pressure microfluidization for 3 times, the colostrum was filtered through 0.22 μm micropores to obtain 4-butylresorcinol nanoliposomes. The mass ratio of 4-butylresorcinol to 3-o-ethylascorbic acid is 1:0.1, the mass ratio of 4-butylresorcinol to ascorbyl tetraisopalmitate is 1:0.1, the mass ratio of 4-butylresorcinol to hydrogenated lecithin is 1:0.8, the mass ratio of 4-butylresorcinol to cholesterol is 1:0.1, the mass ratio of 4-butylresorcinol to caprylic / capric triglyceride is 1:1.8, the mass ratio of 4-butylresorcinol to glycerol is 1:2, the mass ratio of glycerol to water is 1:2.05, and the CAS number of caprylic / capric triglyceride is 65381-09-1.

[0079] Embodiment 2:

[0080] Preparation of DSPE-PEG2000-LPTATH: DSPE-PEG2000-NHS and hexapeptide-2 were dissolved in a mixture of DMF and chloroform to obtain a reaction mixture, N-methylmorpholine was added to the reaction mixture to adjust the pH to 8.0, and the reaction mixture was stirred at 25°C for 24 h under argon protection to obtain a reaction solution, the reaction solution was placed in a 3500 MW dialysis bag, immersed in deionized water for dialyzation, the deionized water was changed every 5 h, and the dialyzate was obtained by 48 h of dialyzation, and the dialyzate was freeze-dried to obtain DSPE-PEG2000-LPTATH. The mass ratio of DSPE-PEG2000-NHS to hexapeptide-2 is 1:0.15, DMF and chloroform are mixed in a volume ratio of 1:1, the amount of DSPE-PEG2000-NHS in the mixed solution of DMF and chloroform is 0.087 g / ml, and DSPE-PEG2000-NHS is the abbreviation of distearoylphosphatidylethanolamine-polyethylene glycol-succinimide ester.

[0081] Preparation of 4-butylresorcinol nanoliposomes: 4-butylresorcinol, 3-o-ethyl ascorbic acid, ascorbyl tetraisopalmitate, hydrogenated lecithin, cholesterol, caprylic / capric triglyceride and DSPE-PEG2000-LPTATH were stirred and mixed to obtain phase A. Glycerol and water were stirred and mixed to obtain phase B. Then phase A was added to phase B and homogenized at 3000 r / min for 2 min to obtain colostrum. Then, the colostrum was subjected to high-pressure microfluidization at 20,000 psi. After repeated homogenization by high-pressure microfluidization for 3 times, the colostrum was filtered through 0.22 μm micropores to obtain 4-butylresorcinol nanoliposomes. The mass ratio of 4-butylresorcinol to 3-o-ethylascorbic acid is 1:0.1, the mass ratio of 4-butylresorcinol to ascorbyl tetraisopalmitate is 1:0.1, the mass ratio of 4-butylresorcinol to hydrogenated lecithin is 1:0.8, the mass ratio of 4-butylresorcinol to cholesterol is 1:0.1, the mass ratio of 4-butylresorcinol to caprylic / capric triglyceride is 1:1.8, the mass ratio of 4-butylresorcinol to DSPE-PEG2000-LPTATH is 1:0.1, the mass ratio of 4-butylresorcinol to glycerol is 1:2, and the mass ratio of glycerol to water is 1:2.

[0082] Embodiment 3:

[0083] The preparation of DSPE-PEG2000-LPTATH is the same as in Example 2.

[0084] Preparation of vanillic acid ester derivatives: p-Methoxybenzylamine was added to a mixed solution of maleic anhydride and acetonitrile, and the mixture was refluxed for 3 hours. After the reaction was completed, the mixture was cooled to 20°C to precipitate a solid, which was filtered out and dried to obtain a crude product. Vanillic acid ester was dissolved in dichloromethane to obtain a mixed solution, and the crude product, 2-methyl-1,3-propanediol, dicyclohexylcarbodiimide and 4-dimethylaminopyridine were added to the mixed solution, and the reaction was stirred at 0°C for 6 hours to obtain a reaction solution, and the reaction solution was concentrated and filtered to obtain a filtrate, and the filtrate was evaporated to obtain a vanillic acid ester derivative. The mass ratio of p-methoxybenzylamine to maleic anhydride is 1:1.03, the amount of maleic anhydride in acetonitrile in the maleic anhydride acetonitrile mixed solution is 0.1 g / ml, the mass ratio of vanillic acid ester to the crude product is 1:1.1, the mass ratio of vanillic acid ester to 2-methyl-1,3-propanediol is 1:0.6, the mass ratio of vanillic acid ester to dicyclohexylcarbodiimide is 1:1.42, and the mass ratio of vanillic acid ester to 4-dimethylaminopyridine is 1:0.042.

[0085] The vanillic acid ester derivatives prepared by the present invention were subjected to a safety assessment through a mouse transdermal toxicity test, in which the vanillic acid ester derivatives were administered to mice once transdermally and the symptoms were observed. The assessment results showed that the experimental mice did not show signs of poisoning, that is, the vanillic acid ester derivatives prepared by the present invention were of a practically non-toxic level.

[0086] Preparation of 4-butylresorcinol nanoliposomes: 4-butylresorcinol, 3-o-ethyl ascorbic acid, ascorbyl tetraisopalmitate, vanillate derivatives, hexadecyl citric acid, hydrogenated lecithin, cholesterol, caprylic / capric triglyceride and DSPE-PEG2000-LPTATH were stirred and mixed to obtain phase A. Glycerol and water were stirred and mixed to obtain phase B. Then phase A was added to phase B and homogenized at 3000 r / min for 2 min to obtain colostrum. Then, the colostrum was subjected to high-pressure microfluidization at 20000 psi. After repeated homogenization by high-pressure microfluidization for 3 times, the colostrum was filtered through 0.22 μm micropores to obtain 4-butylresorcinol nanoliposomes. The mass ratio of 4-butylresorcinol to 3-o-ethylascorbic acid is 1:0.1, the mass ratio of 4-butylresorcinol to ascorbic acid tetraisopalmitate is 1:0.1, the mass ratio of 4-butylresorcinol to vanillate derivative is 1:0.1, the mass ratio of 4-butylresorcinol to hexadecyl citric acid is 1:0.03, the mass ratio of 4-butylresorcinol to hydrogenated lecithin is 1:0.1, and the mass ratio of 4-butylresorcinol to hexadecyl citric acid is 1:0.03. The mass ratio of the amount of 4-butylresorcinol to the amount of cholesterol is 1:0.1, the mass ratio of the amount of 4-butylresorcinol to the amount of caprylic / capric triglyceride is 1:1.8, the mass ratio of the amount of 4-butylresorcinol to the amount of DSPE-PEG2000-LPTATH is 1:0.1, the mass ratio of the amount of 4-butylresorcinol to the amount of glycerol is 1:2, and the mass ratio of the amount of glycerol to the amount of water is 1:2.

[0087] Embodiment 4:

[0088] The preparation of DSPE-PEG2000-LPTATH is the same as in Example 2.

[0089] The preparation of the vanillic acid ester derivative is the same as in Example 3.

[0090] Preparation of modified phospholipids: phospholipids, conjugated linoleic acid ethyl ester and lipase are added to n-hexane to obtain a mixture, the mixture is reacted at 200 r / min and 55°C for 24 hours to obtain a reaction product, the reaction product is centrifuged at 4000 r / min for 10 minutes, then evaporated at 50°C to remove n-hexane, and finally washed with cold acetone 3 times to obtain an insoluble solid, and the insoluble solid is vacuum dried at 50°C to obtain the modified phospholipids. The mass ratio of the amount of phospholipids used to the amount of conjugated linoleic acid ethyl ester used is 1:3, and the mass ratio of the amount of phospholipids used to the amount of lipase used is 1:0.2.

[0091] Preparation of 4-butylresorcinol nanoliposomes: 4-butylresorcinol, 3-o-ethyl ascorbic acid, ascorbyl tetraisopalmitate, vanillate derivatives, hexadecyl citric acid, hydrogenated lecithin, modified phospholipids, cholesterol, caprylic / capric triglyceride and DSPE-PEG2000-LPTATH were stirred and mixed to obtain phase A. Glycerol and water were stirred and mixed to obtain phase B. Then phase A was added to phase B and homogenized at 3000 r / min for 2 min to obtain colostrum. Then, the colostrum was subjected to high-pressure microfluidization at 20000 psi. After repeated homogenization by high-pressure microfluidization for 3 times, the colostrum was filtered through 0.22 μm micropores to obtain 4-butylresorcinol nanoliposomes. The mass ratio of 4-butylresorcinol to 3-o-ethylascorbic acid is 1:0.1, the mass ratio of 4-butylresorcinol to ascorbic acid tetraisopalmitate is 1:0.1, the mass ratio of 4-butylresorcinol to vanillate derivative is 1:0.1, the mass ratio of 4-butylresorcinol to hexadecyl citric acid is 1:0.1, the mass ratio of 4-butylresorcinol to hydrogenated lecithin is 1:0.8, and the mass ratio of 4-butylresorcinol to hexadecyl citric acid is 1:0.1. The mass ratio of phenol to modified phospholipid is 1:0.4, the mass ratio of 4-butylresorcinol to cholesterol is 1:0.1, the mass ratio of 4-butylresorcinol to caprylic / capric triglyceride is 1:1.8, the mass ratio of 4-butylresorcinol to DSPE-PEG2000-LPTATH is 1:0.1, the mass ratio of 4-butylresorcinol to glycerol is 1:2, and the mass ratio of glycerol to water is 1:2.

[0092] Embodiment 5:

[0093] The preparation of DSPE-PEG2000-LPTATH is the same as in Example 2.

[0094] The preparation of the vanillic acid ester derivative is the same as in Example 3.

[0095] The preparation of modified phospholipids is the same as in Example 4.

[0096] Preparation of 4-butylresorcinol nanoliposomes: The preparation of 4-butylresorcinol nanoliposomes in this example is compared with that in Example 4, except that the mass ratio of the amount of 4-butylresorcinol used to the amount of modified phospholipid used is 1:0.6, and the other conditions and parameters are the same as in Example 4.

[0097] Embodiment 6:

[0098] The preparation of DSPE-PEG2000-LPTATH is the same as in Example 2.

[0099] The preparation of the vanillic acid ester derivative is the same as in Example 3.

[0100] The preparation of modified phospholipids is the same as in Example 4.

[0101] Preparation of 4-butylresorcinol nanoliposomes: The preparation of 4-butylresorcinol nanoliposomes in this example is compared with that in Example 4, except that the mass ratio of the amount of 4-butylresorcinol used to the amount of modified phospholipid used is 1:0.2, and the other conditions and parameters are the same as in Example 4.

[0102] Comparative Example 1:

[0103] Preparation of blank liposomes: The preparation of blank liposomes in this example is compared with the preparation of butylresorcinol nanoliposomes in Example 1, except that 4-butylresorcinol is not added in this example, the mass ratio of the amount of glycerol used to the amount of water used is 1:2.55, and other conditions and parameters are the same as in Example 1.

[0104] Comparative Example 2:

[0105] Preparation of targeted blank liposomes: The preparation of targeted blank liposomes in this example is compared with the preparation of butylresorcinol nanoliposomes in Example 2, except that 4-butylresorcinol is not added in this example, the mass ratio of the amount of glycerol used and the amount of water used is 1:2.5, and other conditions and parameters are the same as in Example 2.

[0106] Comparative Example 3:

[0107] The preparation of DSPE-PEG2000-LPTATH is the same as in Example 2.

[0108] Preparation of 4-butylresorcinol nanoliposomes: 4-butylresorcinol, 3-o-ethyl ascorbic acid, ascorbyl tetraisopalmitate, hydrogenated lecithin, cholesterol, caprylic / capric triglyceride and DSPE-PEG2000-LPTATH were dissolved in ethanol, and then the ethanol was evaporated at 35°C to obtain a light yellow film. Then, glycerol and water were added to the light yellow film, and ultrasonic reaction was carried out at 35°C for 30 minutes to obtain 4-butylresorcinol nanoliposomes. The mass ratio of 4-butylresorcinol to 3-o-ethylascorbic acid is 1:0.1, the mass ratio of 4-butylresorcinol to ascorbyl tetraisopalmitate is 1:0.1, the mass ratio of 4-butylresorcinol to hydrogenated lecithin is 1:0.8, the mass ratio of 4-butylresorcinol to cholesterol is 1:0.1, the mass ratio of 4-butylresorcinol to caprylic / capric triglyceride is 1:1.8, the mass ratio of 4-butylresorcinol to DSPE-PEG2000-LPTATH is 1:0.1, the mass ratio of 4-butylresorcinol to glycerol is 1:2, and the mass ratio of glycerol to water is 1:2.

[0109] Comparative Example 4:

[0110] The preparation of DSPE-PEG2000-LPTATH is the same as in Example 2.

[0111] Preparation of 4-butylresorcinol nanoliposomes: 4-butylresorcinol, 3-o-ethyl ascorbic acid, ascorbyl tetraisopalmitate, hydrogenated lecithin, cholesterol, caprylic / capric triglyceride and DSPE-PEG2000-LPTATH were added to ethanol to dissolve to obtain a mixture, and then a mixed medium of glycerol and water was injected into the mixture, and the reaction was stirred for 60 minutes. After the reaction was completed, the ethanol was evaporated to obtain 4-butylresorcinol nanoliposomes. The mass ratio of 4-butylresorcinol to 3-o-ethylascorbic acid is 1:0.1, the mass ratio of 4-butylresorcinol to ascorbyl tetraisopalmitate is 1:0.1, the mass ratio of 4-butylresorcinol to hydrogenated lecithin is 1:0.8, the mass ratio of 4-butylresorcinol to cholesterol is 1:0.1, the mass ratio of 4-butylresorcinol to caprylic / capric triglyceride is 1:1.8, the mass ratio of 4-butylresorcinol to DSPE-PEG2000-LPTATH is 1:0.1, the mass ratio of 4-butylresorcinol to glycerol is 1:2, and the mass ratio of glycerol to water is 1:2.

[0112] Comparative Example 5:

[0113] The preparation of DSPE-PEG2000-LPTATH is the same as in Example 2.

[0114] The preparation of the vanillic acid ester derivative is the same as in Example 3.

[0115] Preparation of 4-butylresorcinol nanoliposomes: The preparation of 4-butylresorcinol nanoliposomes in this comparative example is compared with that in Example 3, except that hexadecyl citric acid is not used in this example, and other conditions and parameters are the same as in Example 1.

[0116] Comparative Example 6:

[0117] The preparation of DSPE-PEG2000-LPTATH is the same as in Example 2.

[0118] Preparation of 4-butylresorcinol nanoliposomes: The preparation of 4-butylresorcinol nanoliposomes in this comparative example is compared with that in Example 3, except that no vanillate derivatives are used in this example, and other conditions and parameters are the same as in Example 1.

[0119] Experimental Example 1:

[0120] Particle size, PDI and encapsulation efficiency test: the liposomes prepared in Examples 1-2 and Comparative Examples 1-4 were diluted 150 times with water, and the particle size and PDI were measured. After dilution with water, the supernatant was taken after centrifugation for 10 min to measure the free active substance concentration C1. Take the liposomes prepared in Examples 1-2 and Comparative Examples 1-4, add methanol and ultrasonically break the emulsion, and measure the total drug concentration C2. The encapsulation efficiency was calculated according to the formula encapsulation efficiency %=1-C1 / C2×100%. The liposomes include 4-butylresorcinol nanoliposomes prepared in Examples 1-2 and Comparative Examples 3-4, blank liposomes prepared in Comparative Example 1, and targeted blank liposomes prepared in Comparative Example 2.

[0121] Table 1 Particle size, PDI and encapsulation efficiency test results

[0122]

[0123] The results are shown in Table 1. The particle size of the liposomes prepared in Examples 1-2 and Comparative Examples 1-2 is between 120-130 nm, and the PDI is relatively low. The particle size and PDI of the liposomes prepared in Example 2 are smaller than those in Comparative Examples 3-4, indicating that high-pressure microjetting can make the particle size of the liposomes smaller and the particle size distribution more uniform due to its huge energy input compared to the thin film method and the injection method; the encapsulation efficiency of the liposomes prepared in Example 2 is greater than that of Comparative Examples 3-4, indicating that the use of high-pressure microjetting can reduce the loss of active substances and improve the overall encapsulation efficiency.

[0124] Experimental Example 2:

[0125] To test the targeting of liposomes, mouse melanoma B16 cells were cultured to the logarithmic growth phase and the concentration of the cell suspension was adjusted to 1×10 5 Each group was inoculated with equal amounts of cells / mL and incubated at 37°C and 5% CO 2 After 24 hours of culture, the blank group was added with culture medium, the model group was added with culture medium containing α-MSH, and the sample group was added with culture medium containing α-MSH and liposomes, and the cell lysis solution was obtained after 24 hours of culture. The absorbance of each group at 450 nm was measured using a cAMP ELISA kit, and cAMP was calculated based on the absorbance. The amount of α-MSH in the culture medium was 0.33 mg / ml, the mass ratio of the amount of liposomes used to the amount of culture medium used was 1:50, the volume ratio of the amount of culture medium used to the amount of cell suspension used was 1:1, and the liposomes included the 4-butylresorcinol nanoliposomes prepared in Example 1-2, the blank liposomes prepared in Comparative Example 1, and the targeted blank liposomes prepared in Comparative Example 2.

[0126] The results of the test are as follows Figure 1 As shown, in the model group, after induction by α-MSH, the cAMP content was significantly increased, proving that the model was successfully established. The cAMP content measured in the group containing the liposomes prepared in Example 2 was lower than that in the group containing Example 1; the cAMP content measured in the group containing the liposomes prepared in Comparative Example 2 was lower than that in the group containing Comparative Example 1, indicating that the liposomes prepared by using DSPE-PEG2000-LPTATH have targeting properties.

[0127] Experimental Example 3:

[0128] To determine the effect of liposomes on the relative activity of tyrosinase, mouse melanoma B16 cells were cultured to the logarithmic growth phase and the concentration of the cell suspension was adjusted to 1×10 5 / mL, inoculate in equal amounts, and place at 37°C, 5% CO 2 After 24 h of culture, all groups except the blank group were placed in 30 J / cm 2Irradiation was performed under UVB light source. After irradiation, the waste liquid was discarded, the blank group was added with culture medium, the model group was added with culture medium containing α-MSH, and the sample group was added with culture medium containing α-MSH and liposomes. After culturing for 24 h, the supernatant was discarded, washed with PBS solution for 3 times, and then 1% (v / v) Triton X-100 solution was added. After freezing at -80℃ for 40 min, the cell lysate was thawed to obtain the cell lysate, the cell lysate was centrifuged to obtain the supernatant, 0.1% (w / v) L-dopa solution was added, and it was placed at 37℃ for 2 h. The absorbance was measured at 475 nm to calculate the tyrosinase activity. The amount of α-MSH in the culture medium is 0.33 mg / ml, the mass ratio of the amount of liposomes used to the amount of culture medium used is 1:50, the volume ratio of the amount of culture medium used to the amount of cell suspension used is 1:1, the volume ratio of the amount of 1% (v / v) Triton X-100 solution used to the amount of cell suspension used is 1:1, the volume ratio of the amount of 0.1% (w / v) levodopa solution used to the amount of cell suspension used is 1:1, and the liposomes include 4-butylresorcinol nanoliposomes prepared in Examples 1-6 and Comparative Examples 5-6, blank liposomes prepared in Comparative Example 1, and targeted blank liposomes prepared in Comparative Example 2.

[0129] The results of the test are as follows Figure 2 As shown, the relative activity of tyrosinase in the model group reached 159.32% under the induction of UVB light source, proving that the modeling was successful. The relative activity of tyrosinase in the liposome sample group prepared in Example 1 was lower than that in Comparative Example 1, indicating that the use of 4-butylresorcinol has a good effect on the inhibition of the relative activity of tyrosinase; the relative activity of tyrosinase in the liposome sample group prepared in Example 2 was lower than that in Example 1, indicating that the use of DSPE-PEG2000-LPTATH can further reduce the relative activity of tyrosinase; the relative activity of tyrosinase in the liposome sample group prepared in Example 3 was lower than that in Example 2, indicating that the use of vanillic acid ester derivatives and hexadecyl citric acid can enhance the regulatory effect on tyrosinase. , thereby inhibiting the relative activity of tyrosinase; the relative activity of tyrosinase in the liposome sample group prepared in Example 4 is lower than that in Example 3, indicating that on the basis of using vanillic acid ester derivatives and hexadecyl citric acid, the use of modified phospholipids can further improve the inhibitory effect of liposomes on the relative activity of tyrosinase; the relative activity of tyrosinase in the liposome sample group prepared in Example 4 is lower than that in Comparative Examples 5-6, indicating that vanillic acid ester derivatives and hexadecyl citric acid need to be used at the same time, and the use of any one of vanillic acid ester derivatives and hexadecyl citric acid alone has no obvious effect on the inhibition of the relative activity of tyrosinase.

[0130] Experimental Example 4:

[0131] To determine the effect of liposomes on melanin production, mouse melanoma B16 cells were cultured to the logarithmic growth phase and the concentration of the cell suspension was adjusted to 1×10 5 / mL, inoculate in equal amounts, and place at 37°C, 5% CO 2 After 24 h of culture, all groups except the blank group were placed in 30 J / cm 2 Irradiation was performed under UVB light source. After irradiation, the waste liquid was discarded, the blank group was added with culture medium, the model group was added with culture medium containing α-MSH, and the sample group was added with culture medium containing α-MSH and liposomes. After continuing to culture for 24 h, the supernatant was discarded, washed with PBS solution 3 times, and then 1.0 mol / L NaOH solution (containing 10% DMSO) was added, and the cells were fully lysed by heating in a water bath at 80°C, and the supernatant was taken by centrifugation, and the absorbance was measured at a wavelength of 405 nm, and the relative content of melanin was calculated. The amount of α-MSH in the culture medium is 0.33 mg / ml, the mass ratio of the amount of liposomes used to the amount of culture medium used is 1:50, the volume ratio of the amount of culture medium used to the amount of cell suspension used is 1:1, the volume ratio of the amount of NaOH solution used to the amount of cell suspension used is 1:1, and the liposomes include 4-butylresorcinol nanoliposomes prepared in Examples 1-6 and Comparative Examples 5-6, blank liposomes prepared in Comparative Example 1, and targeted blank liposomes prepared in Comparative Example 2.

[0132] The results of the test are as follows Figure 3 As shown, the relative content of melanin in the model group reached 132.19% under the induction of UVB light source, proving that the model was successful. The relative content of melanin in the liposome sample group prepared in Example 1 is lower than that in Comparative Example 1, indicating that the use of 4-butylresorcinol can inhibit the formation of melanin, thereby reducing the relative content of melanin; the relative content of melanin in the liposome sample group prepared in Example 2 is lower than that in Example 1, indicating that the use of DSPE-PEG2000-LPTATH can further reduce the relative content of melanin; the relative content of melanin in the liposome sample group prepared in Example 3 is lower than that in Example 2, indicating that the use of vanillic acid ester derivatives and hexadecyl citric acid can enhance the inhibitory effect on melanin formation; the relative content of melanin in the liposome sample group prepared in Example 4 is lower than that in Example 3, indicating that on the basis of using vanillic acid ester derivatives and hexadecyl citric acid, the use of modified phospholipids can further inhibit melanin formation; the relative content of melanin in the liposome sample group prepared in Example 4 is lower than that in Comparative Examples 5-6, indicating that vanillic acid ester derivatives and hexadecyl citric acid need to be used simultaneously, and the use of any one of the vanillic acid ester derivatives and hexadecyl citric acid alone has no obvious effect on reducing the relative content of melanin.

[0133] Experimental Example 5:

[0134] The chromaticity growth rate was determined by storing the liposomes prepared in Examples 1-6 and Comparative Examples 1-2 and 5-6 at 40° C. for 28 days, comparing the chromaticity with the initial chromaticity, and calculating the chromaticity growth rate. The liposomes included 4-butylresorcinol nanoliposomes prepared in Examples 1-6 and Comparative Examples 5-6, blank liposomes prepared in Comparative Example 1, and targeted blank liposomes prepared in Comparative Example 2.

[0135] Table 2 Chromaticity growth rate measurement results

[0136]

[0137] The results are shown in Table 2. The chromaticity growth rate of the liposomes prepared in Example 1 is lower than that in Comparative Example 1, indicating that the use of 4-butylresorcinol enhances the stability of the liposomes, thereby reducing the chromaticity growth rate; the chromaticity growth rate of the liposomes prepared in Example 2 is lower than that in Example 1, indicating that the use of DSPE-PEG2000-LPTATH can further delay the chromaticity change and reduce the chromaticity growth rate; the chromaticity growth rate of the liposomes prepared in Example 3 is lower than that in Example 2, indicating that the use of vanillic acid ester derivatives and hexadecyl citric acid can better prevent the discoloration of the liposomes and reduce the chromaticity growth rate; the chromaticity growth rate of the liposomes prepared in Example 4 is lower than that in Example 3, indicating that on the basis of using vanillic acid ester derivatives and hexadecyl citric acid, the use of modified phospholipids can further maintain the stability of the entire liposome system and reduce the chromaticity growth rate; the chromaticity growth rate of the liposomes prepared in Example 4 is lower than that of Comparative Examples 5-6, indicating that vanillic acid ester derivatives and hexadecyl citric acid need to be used simultaneously, and the use of any one of the vanillic acid ester derivatives and hexadecyl citric acid alone has no obvious effect on reducing the chromaticity growth rate.

[0138] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0139] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A nanoliposome, comprising: Active substances, antioxidants, carriers and adjuvants; the antioxidants are vanillic acid ester derivatives and hexadecyl citric acid; The preparation method of the vanillic acid ester derivative is specifically as follows: adding p-methoxybenzylamine to a mixed solution of maleic anhydride and acetonitrile for reaction, cooling to 5-25°C after the reaction to precipitate a solid, filtering and drying the solid to obtain a crude product; mixing vanillic acid ester with dichloromethane to obtain a mixed solution, adding the crude product, 2-methyl-1,3-propanediol, dicyclohexylcarbodiimide and 4-dimethylaminopyridine to the mixed solution, the temperature is 0-5°C, the reaction time is 5-8h, and after the reaction, filtering and evaporating to obtain the vanillic acid ester derivative; The mass ratio of the amount of p-methoxybenzylamine to the amount of maleic anhydride is 1:0.8-1.2, the amount of maleic anhydride in acetonitrile in the maleic anhydride acetonitrile mixed solution is 0.08-0.13 g / ml, the mass ratio of the amount of vanillic acid ester to the amount of the crude product is 1:0.7-1.5, the mass ratio of the amount of vanillic acid ester to the amount of 2-methyl-1,3-propanediol is 1:0.4-0.9, the mass ratio of the amount of vanillic acid ester to the amount of dicyclohexylcarbodiimide is 1:1-2, and the mass ratio of the amount of vanillic acid ester to the amount of 4-dimethylaminopyridine is 1:0.02-0.1; The active substance is 4-butylresorcinol; The carrier further comprises phospholipid, cholesterol and DSPE-PEG2000-LPTATH; the phospholipid is at least one of soybean lecithin, sunflower lecithin, hydrogenated lecithin, phosphatidylcholine and hydrogenated phosphatidylcholine; DSPE-PEG2000-LPTATH is obtained by covalently bonding DSPE-PEG2000-NHS and hexapeptide-2; The mass ratio of the active substance to the antioxidant is 1:0.1-0.5, the mass ratio to the carrier is 1:0.3-1.8, and the mass ratio to the auxiliary is 1:4.5-16.

2. A nanoliposome according to claim 1, characterized in that: The auxiliary agent comprises a lipid auxiliary agent and a water phase auxiliary agent, wherein the lipid auxiliary agent is caprylic acid / capric acid triglyceride, and the water phase auxiliary agent is glycerol and water.

3. A method for preparing the nanoliposome according to claim 1, comprising: The active substance, antioxidant, carrier and lipid auxiliary agent are mixed to obtain phase A, and the aqueous phase auxiliary agents are mixed to obtain phase B; Then, phase A is added to phase B for homogenization, and then subjected to high-pressure microfluidization treatment to obtain nanoliposomes.

4. The method for preparing nanoliposomes according to claim 3, characterized in that: The active substance is 4-butylresorcinol, the antioxidant is at least one of 3-o-ethyl ascorbic acid, ascorbyl palmitate, vanillate derivatives and hexadecyl citric acid, and the mass ratio of the active substance to the antioxidant is 1:0.1-0.

5.

5. The method for preparing nanoliposomes according to claim 3, characterized in that: The carrier comprises phospholipid, cholesterol and DSPE-PEG2000-LPTATH, wherein the phospholipid is at least one of soybean lecithin, sunflower lecithin, hydrogenated lecithin, phosphatidylcholine and hydrogenated phosphatidylcholine; the lipid auxiliary agent is caprylic acid / capric acid triglyceride; and the water phase auxiliary agent comprises glycerol and water.

6. The method for preparing nanoliposomes according to claim 3, characterized in that: The homogenization speed of the phase A and the phase B is 2500-4000 r / min, the homogenization time is 1.5-5 min, and the pressure of the high-pressure microjet is 18000-30000psi.

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