Targeted mitochondria curcumin nano-emulsion as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a targeted mitochondria curcumin nano-emulsion as well as a preparation method and application thereof. Comprising the following steps: dissolving triphenylphosphine and a carrier in a solvent, and reacting in the presence of a condensing agent and / or a catalyst to obtain a carrier-TPP which is zein-TPP or hyaluronic acid-TPP; zein-TPP or zein is taken as a core, curcumin is loaded, hyaluronic acid or hyaluronic acid-TPP is taken as a shell, and the targeting mitochondria curcumin nano-emulsion is obtained through crosslinking. When the zein-TPP is taken as the core, hyaluronic acid is taken as the shell, and when the zein is taken as the core, hyaluronic acid-TPP is taken as the shell. According to the invention, TPP is utilized to modify a carrier, curcumin is embedded into a zein kernel and is coated by sodium hyaluronate anions, curcumin can be delivered to mitochondria in a targeted manner, accumulation concentration of curcumin in mitochondria is enhanced, and mitochondria aging resistance is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics, and particularly relates to a mitochondria-targeted curcumin nanoemulsion and a preparation method and application thereof. Background Art

[0002] Skin aging is divided into endogenous aging caused by aging and exogenous aging caused by factors such as sun exposure, environmental pollution, smoking, and lifestyle. Its key feature is the direct consequence of mitochondrial dysfunction. These aging phenotypes include wrinkles, graying and hair loss, uneven pigmentation, and slow wound healing. When mitochondrial function weakens, the energy metabolism of skin cells will be impaired, resulting in slower skin turnover and reduced cell synthesis and secretion of collagen. Collagen is an important component for maintaining skin elasticity and firmness. Its reduction will cause the skin to become dull, dull, less elastic, and show signs of aging such as fine lines and wrinkles.

[0003] In addition, mitochondria produce reactive oxygen species (ROS) in the process of generating energy. When the amount of reactive oxygen species produced is high, mitochondrial DNA will be damaged by oxidation, further aggravating skin aging. Therefore, maintaining the health and function of mitochondria is crucial to delaying skin aging.

[0004] Turmeric is a commonly used traditional Chinese medicine. It comes from the dried rhizome of the ginger plant Curcuma longa. It is an herb with a long history of application in India, China, Southeast Asia and other countries. In the field of skin care, curcumin has the following effects: anti-inflammatory effect, which can reduce the inflammatory response of sensitive skin and inflammatory skin diseases; antioxidant, which can neutralize free radicals to prevent skin aging; and anti-photoaging, which can reduce the damage of ultraviolet rays to the skin. Curcumin can activate the autophagy process, which helps to remove damaged proteins and aging organelles. However, there are problems in the application of curcumin, such as poor photothermal stability, low bioavailability, and easy decomposition under oxidation. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a mitochondrial targeted curcumin nanoemulsion and its preparation method and application. The present invention uses triphenylphosphine (TPP) to modify the shell carrier system, prepares a targeted carrier, and encapsulates curcumin by a layer-by-layer self-assembly method; first, the hydrophobic curcumin is embedded in the corn protein kernel, and the shell is further coated with a sodium hyaluronate anion shell. The nanoemulsion can deliver curcumin to the mitochondria, enhance the accumulation concentration of curcumin in the mitochondria, thereby realizing mitochondrial anti-aging at the root and achieving precise skin care effects.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] One of the purposes of the present invention is to provide a method for preparing a mitochondrial-targeted curcumin nanoemulsion, comprising the following steps:

[0008] Dissolve triphenylphosphine and a carrier in a solvent, and under the action of a condensing agent and / or a catalyst, triphenylphosphine and the carrier undergo an amidation reaction or an esterification reaction to obtain a carrier-TPP; wherein the carrier-TPP is zein-TPP or hyaluronic acid-TPP.

[0009] Zein-TPP or zein is used as the core, curcumin is embedded and loaded, and then hyaluronic acid or hyaluronic acid-TPP is used as the shell, and the core and shell are cross-linked to obtain mitochondrial curcumin nanoemulsion; when zein-TPP is the core, hyaluronic acid is used as the shell, and when zein is the core, hyaluronic acid-TPP is used as the shell.

[0010] Further, when the carrier-TPP is zein-TPP, the preparation method comprises the following steps:

[0011] The triphenylphosphine and zein are dissolved in a solvent, and under the action of a condensing agent, the zein and the triphenylphosphine undergo an amidation reaction to obtain zein-TPP.

[0012] Among them, the mass ratio of zein, triphenylphosphine and condensation agent is 6-22:1:2-13, the second solvent is dimethyl sulfoxide; the condensation agent is a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 1.5-10:1, the temperature of the amidation reaction is 20°C-30°C, and the reaction time is 15h-24h.

[0013] Further, when the carrier-TPP is hyaluronic acid-TPP, the preparation method comprises the following steps:

[0014] Dissolve triphenylphosphine and hyaluronic acid in a solvent, and under the action of a condensing agent and a catalyst, hyaluronic acid and triphenylphosphine undergo an esterification reaction to obtain hyaluronic acid-TPP.

[0015] Among them, the mass ratio of hyaluronic acid, triphenylphosphine, condensation agent and catalyst is 6-22:1:1-9:0.3-3; the second solvent is water; the condensation agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, the catalyst is 4-dimethylaminopyridine, the temperature of the amidation reaction is 20°C-30°C, and the reaction time is 15h-24h.

[0016] Furthermore, when the carrier-TPP is zein-TPP, the preparation method of the mitochondrial-targeted curcumin nanoemulsion comprises the following steps:

[0017] dissolving zein-TPP and cholesterol or lecithin in a solvent to form a mixed solution A;

[0018] Curcumin solution is added to mixed solution A to embed curcumin into carrier-TPP to obtain mixed solution B, and then hyaluronic acid solution is added to mixed solution B. Zein-TPP is used as the core and hyaluronic acid is used as the shell. Hyaluronic acid and zein-TPP are cross-linked to obtain mitochondrial curcumin nanoemulsion.

[0019] Furthermore, the mass ratio of cholesterol or lecithin to zein-TPP is 1:1-2, the concentration of zein-TPP in the mixed solution A is 5 mg / ml-20 mg / ml, the mass ratio of zein-TPP to curcumin in the curcumin solution is 10:3-30, and the concentration of the curcumin solution is 3 mg / ml-12 mg / ml; the volume ratio of the mixed solution B to the hyaluronic acid solution is 2:30, and the concentration of the hyaluronic acid solution is 5 mg / ml-10 mg / ml.

[0020] Furthermore, when the carrier-TPP is hyaluronic acid-TPP, the preparation method of the mitochondrial targeted curcumin nanoemulsion comprises the following steps:

[0021] dissolving zein and cholesterol or lecithin in a solvent to form a mixed solution C;

[0022] The curcumin solution is added to the mixed solution C, so that the curcumin is embedded and loaded in the zein to form a mixed solution D, and then the hyaluronic acid-TPP solution is added to the mixed solution D, with the zein as the core and the hyaluronic acid-TPP as the shell, and the hyaluronic acid-TPP and the zein are cross-linked to obtain a mitochondrial-targeted curcumin nanoemulsion.

[0023] Furthermore, the mass ratio of cholesterol or lecithin to zein is 1:1-2, the concentration of zein in the mixed solution C is 5 mg / ml-20 mg / ml, the mass ratio of zein to curcumin in the curcumin solution is 10:3-30, the concentration of the curcumin solution is 3 mg / ml-12 mg / ml, the volume ratio of the mixed solution D to the hyaluronic acid-TPP solution is 2:1, and the concentration of the hyaluronic acid-TPP solution is 5 mg / ml-10 mg / mlmg / ml.

[0024] Furthermore, the cross-linking reaction temperature is 20°C to 60°C, and the time is 0.5h to 3h.

[0025] The second object of the present invention is to provide the mitochondria-targeted curcumin nanoemulsion obtained by the above preparation method.

[0026] The third object of the present invention is to provide the use of the above-mentioned mitochondria-targeted curcumin nanoemulsion in the preparation of cosmetics for delaying skin aging.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The invention provides a method for preparing a mitochondria-targeted curcumin nanoemulsion. The method comprises the following steps: using zein-TPP or zein as an outer shell carrier to encapsulate hydrophobic curcumin, embedding the hydrophobic curcumin into a zein kernel, and then using hyaluronic acid or hyaluronic acid-TPP as a shell for cross-linking. When the carrier-TPP is the core, the hyaluronic acid is used as the shell, and when the zein is the core, the carrier-TPP is used as the shell. The curcumin is encapsulated by a layer-by-layer self-assembly method, the hydrophobic curcumin is embedded into the zein kernel, the outer shell is further coated with a sodium hyaluronate anion shell, and triphenylphosphine (TPP) has a specific small molecule compound that is combined with a mitochondrial membrane and enters the interior of the mitochondria, thereby regulating the function of the cell. The prepared nanoemulsion can deliver curcumin to the mitochondria, enhance the accumulation concentration of curcumin in the mitochondria, thereby realizing mitochondrial anti-aging at the root and achieving a precise skin care effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a microscopic morphology of the mitochondrial targeted curcumin nanoemulsion prepared in Example 1 of the present invention.

[0030] Figure 2 This is the particle size distribution diagram of the mitochondrial targeted curcumin nanoemulsion prepared in Example 2 of the present invention.

[0031] Figure 3 This is a microscopic observation result of the targeted fluorescence image of the mitochondrial targeted curcumin nanoemulsion prepared in Example 1 of the present invention.

[0032] Figure 4 This is a bar graph of the targeting test results of the mitochondrial targeted curcumin nanoemulsion prepared in Example 1 of the present invention.

[0033] Figure 5 This is a bar graph of the cell viability test results of the mitochondrial targeted curcumin nanoemulsion prepared in Example 1 of the present invention.

[0034] Figure 6 This is a microscopic observation result of ROS fluorescence image of mitochondria-targeted curcumin nanoemulsion prepared in Example 1 of the present invention.

[0035] Figure 7 This is a bar graph of the ROS content test results of the mitochondrial targeted curcumin nanoemulsion prepared in Example 1. DETAILED DESCRIPTION

[0036] 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.

[0037] It should be noted that the professional terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, the various raw materials, reagents, instruments and equipment used in the following embodiments of the present invention can be purchased from the market or prepared by existing methods.

[0038] Turmeric is a commonly used traditional Chinese medicine. It comes from the dried rhizome of the ginger plant Curcuma longa. It is an herb with a long history of application in India, China, Southeast Asia and other countries. In the field of skin care, curcumin has the following effects: anti-inflammatory effect, which can reduce the inflammatory response of sensitive skin and inflammatory skin diseases; antioxidant, which can neutralize free radicals to prevent skin aging; and anti-photoaging, which can reduce the damage of ultraviolet rays to the skin. Curcumin can activate the autophagy process, which helps to remove damaged proteins and aging organelles.

[0039] However, curcumin has problems such as poor photothermal stability, low bioavailability, and easy decomposition under oxidation. Therefore, how to encapsulate curcumin into nanocarriers, improve its solubility and stability, and deliver it to the mitochondria of target skin cells is an urgent problem to be solved in order to achieve skin anti-aging.

[0040] Based on the above problems, the present invention provides a method for preparing a mitochondrial targeted curcumin nanoemulsion, comprising the following steps:

[0041] Dissolve triphenylphosphine and a carrier in a solvent, and under the action of a condensing agent and / or a catalyst, triphenylphosphine and the carrier undergo an amidation reaction or an esterification reaction to obtain a carrier-TPP; wherein the carrier-TPP is zein-TPP or hyaluronic acid-TPP.

[0042] Zein-TPP or zein is used as the core, curcumin is embedded and loaded, and then hyaluronic acid or hyaluronic acid-TPP is used as the shell, and the core and shell are cross-linked to obtain mitochondrial curcumin nanoemulsion; when zein-TPP is the core, hyaluronic acid is used as the shell, and when zein is the core, hyaluronic acid-TPP is used as the shell.

[0043] It should be noted that the purpose of the present invention is that curcumin is hydrophobic, has poor stability and low absorption rate, and is intended to be targeted to mitochondria. Based on this, zein-TPP or zein is used as an outer shell carrier to encapsulate the hydrophobic curcumin, embed the hydrophobic curcumin into the zein kernel, and then use hyaluronic acid or hyaluronic acid-TPP as a shell for cross-linking, wherein when the carrier-TPP is the core, the hyaluronic acid is used as the shell, and when the zein is the core, the carrier-TPP is used as the shell, and the curcumin is encapsulated by a layer-by-layer self-assembly method. The hydrophobic curcumin is embedded into the kernel of corn protein, and the outer shell is further coated with a sodium hyaluronate anion shell. Triphenylphosphine (TPP) has a specific small molecule compound that binds to the mitochondrial membrane, allowing more curcumin to accumulate in the mitochondria and enter the interior of the mitochondria, thereby regulating the function of cells. The prepared nanoemulsion can deliver curcumin to the mitochondria, and hyaluronic acid and triphenylphosphine are used to help the active ingredients better target the mitochondria, thereby enhancing the accumulation concentration of curcumin in the mitochondria, thereby achieving mitochondrial anti-aging at the root and achieving precise skin care effects.

[0044] In some embodiments, when the carrier-TPP is zein-TPP, the preparation method comprises the following steps:

[0045] The triphenylphosphine and zein are dissolved in a solvent, and under the action of a condensing agent, the zein and the triphenylphosphine undergo an amidation reaction to obtain zein-TPP.

[0046] The mass ratio of zein, triphenylphosphine and condensation agent is 6-22:1:2-13, the second solvent is dimethyl sulfoxide; the condensation agent is a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 1.5-10:1, the temperature of the amidation reaction is 20°C-30°C, and the reaction time is 15h-24h.

[0047] It should be noted that when the carrier is zein, triphenylphosphine is directly grafted to zein. The phenylphosphine molecule contains a phosphorus atom and three phenyl groups, wherein the phenyl groups are lipophilic and can interact with the hydrophobic regions in the mitochondrial membrane, thereby making the triphenylphosphine mainly enriched in the mitochondria in the cell. Then, when zein-TPP is used as the core and hyaluronic acid is used as the shell, core-shell cross-linking is performed to wrap curcumin through a layer-by-layer self-assembly method, thereby embedding the hydrophobic curcumin into the zein core.

[0048] In some embodiments, when the carrier-TPP is hyaluronic acid-TPP, the preparation method comprises the following steps:

[0049] Dissolve triphenylphosphine and hyaluronic acid in a solvent, and under the action of a condensing agent and a catalyst, hyaluronic acid and triphenylphosphine undergo an esterification reaction to obtain hyaluronic acid-TPP.

[0050] Among them, the mass ratio of hyaluronic acid, triphenylphosphine, condensation agent and catalyst is 6-22:1:1-9:0.3-3; the second solvent is water; the condensation agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, the catalyst is 4-dimethylaminopyridine, the temperature of the amidation reaction is 20°C-30°C, and the reaction time is 15h-24h.

[0051] It should be noted that when the carrier is hyaluronic acid, the hydrophobic curcumin is directly encapsulated by zein, and the hydrophobic curcumin is embedded in the zein kernel to form a core, and triphenylphosphine is grafted with hyaluronic acid to form hyaluronic acid-TPP as a shell, and core-shell cross-linking is performed to encapsulate curcumin through a layer-by-layer self-assembly method, and the hydrophobic curcumin is embedded in the zein kernel.

[0052] In some embodiments, when the carrier-TPP is zein-TPP, the method for preparing the mitochondrial-targeted curcumin nanoemulsion comprises the following steps:

[0053] dissolving zein-TPP and cholesterol or lecithin in a solvent to form a mixed solution A;

[0054] Curcumin solution is added to mixed solution A to embed curcumin into carrier-TPP to obtain mixed solution B, and then hyaluronic acid solution is added to mixed solution B. Zein-TPP is used as the core and hyaluronic acid is used as the shell. Hyaluronic acid and zein-TPP are cross-linked to obtain mitochondrial curcumin nanoemulsion.

[0055] In some embodiments, the mass ratio of cholesterol or lecithin to zein-TPP is 1:1-2, the concentration of zein-TPP in mixed solution A is 5 mg / ml-20 mg / ml, the mass ratio of zein-TPP to curcumin in the curcumin solution is 10:3-30, and the concentration of the curcumin solution is 3 mg / ml-12 mg / ml; the volume ratio of mixed solution B to hyaluronic acid solution is 2:30, and the concentration of the hyaluronic acid solution is 5 mg / ml-10 mg / ml.

[0056] In some embodiments, when the carrier-TPP is hyaluronic acid-TPP, the method for preparing the mitochondrial targeted curcumin nanoemulsion comprises the following steps:

[0057] dissolving zein and cholesterol or lecithin in a solvent to form a mixed solution C;

[0058] The curcumin solution is added to the mixed solution C, so that the curcumin is embedded and loaded in the zein to form a mixed solution D, and then the hyaluronic acid-TPP solution is added to the mixed solution D, with the zein as the core and the hyaluronic acid-TPP as the shell, and the hyaluronic acid-TPP and the zein are cross-linked to obtain a mitochondrial-targeted curcumin nanoemulsion.

[0059] In some embodiments, the mass ratio of cholesterol or lecithin to zein is 1:1-2, the concentration of zein in the mixed solution C is 5 mg / ml-20 mg / ml, the mass ratio of zein to curcumin in the curcumin solution is 10:3-30, the concentration of the curcumin solution is 3 mg / ml-12 mg / ml, the volume ratio of the mixed solution D to the hyaluronic acid-TPP solution is 2:1, and the concentration of the hyaluronic acid-TPP solution is 5 mg / ml-10 mg / mlmg / ml.

[0060] It should be noted that the cholesterol or lecithin used in the present invention is to assist in stabilizing the zein-TPP or zein solution.

[0061] In some embodiments, the cross-linking reaction is carried out at a temperature of 20° C. to 60° C. and for a time of 0.5 h to 3 h.

[0062] The mitochondrial targeted nanoemulsion provided by the present invention has good stability, multi-target and multi-mechanism anti-aging and anti-oxidation, synergistic synergy, and improves the anti-aging effect. At the same time, the nanoparticles promote curcumin to penetrate the skin barrier into the deep tissue of the skin, increase the effective ingredient curcumin to enter cells and mitochondria, thereby significantly improving the bioavailability of the effective ingredient and enhancing its anti-aging and anti-oxidation effects.

[0063] The invention is further described below through specific examples.

[0064] Example 1

[0065] A method for preparing a mitochondrial-targeted curcumin nanoemulsion comprises the following steps:

[0066] S1. Preparation of zein-TPP: 100 mg of triphenylphosphine, 2 g of zein, 400 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 100 mg of N-hydroxysuccinimide were dissolved in 100 ml of dimethyl sulfoxide, and amidation reaction was carried out at room temperature for 15 h. Zein-TPP was obtained by purification by dialysis.

[0067] S2. Weigh 10 mg of zein-TPP and 5 mg of cholesterol, dissolve in 1 mL of 80% ethanol, and dissolve by ultrasonication to form a mixed solution 1; weigh 12 mg of curcumin, dissolve in 1 mL of ethanol, and dissolve to form a mixed solution 2; weigh 150 mg of hyaluronic acid, dissolve in 30 mL of deionized water, and dissolve by stirring at room temperature to form a mixed solution 3.

[0068] S3. Take 1 mL of mixed solution 2 and slowly drip it into 1 mL of mixed solution 1. Stir and react at room temperature for 60 min to form mixed solution 4.

[0069] S4. Take 1 mL of mixed solution 4, slowly drip it into 30 mL of mixed solution 3, stir and react at room temperature for 3 h, and obtain mitochondrial targeted curcumin nanoemulsion.

[0070] Example 2

[0071] A method for preparing a mitochondrial-targeted curcumin nanoemulsion comprises the following steps:

[0072] S1. Preparation of hyaluronic acid-TPP: 100 mg of triphenylphosphine, 2 g of hyaluronic acid, 400 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 100 mg of 4-dimethylaminopyridine were dissolved in 100 ml of water, and an esterification reaction was carried out at room temperature for 15 h. Hyaluronic acid-TPP was obtained by purification by dialysis.

[0073] S2. Weigh 10 mg of zein and 5 mg of cholesterol and dissolve them in 1 mL of 80% ethanol, and dissolve them by ultrasonication to form a mixed solution 1; weigh 12 mg of curcumin and dissolve them in 1 mL of ethanol to form a mixed solution 2; weigh 150 mg of hyaluronic acid-TPP and dissolve them in 30 mL of deionized water, and dissolve them by stirring at room temperature to form a mixed solution 3.

[0074] S3. Take 1 mL of mixed solution 2 and slowly drip it into 1 mL of mixed solution 1. Stir and react at room temperature for 60 min to form mixed solution 4.

[0075] S4. Take 1 mL of mixed solution 4, slowly drip it into 30 mL of mixed solution 3, and stir the mixture at 30° C. for 3 h to obtain mitochondrial targeted curcumin nanoemulsion.

[0076] Example 3

[0077] A method for preparing a mitochondrial-targeted curcumin nanoemulsion comprises the following steps:

[0078] S1. Preparation of hyaluronic acid-TPP: 100 mg of triphenylphosphine, 2 g of hyaluronic acid, 400 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 100 mg of 4-dimethylaminopyridine were dissolved in 100 ml of water, and an esterification reaction was carried out at room temperature for 15 h. Hyaluronic acid-TPP was obtained by purification by dialysis.

[0079] S2. Weigh 10 mg of zein and 5 mg of cholesterol and dissolve them in 1 mL of 80% ethanol, and dissolve them by ultrasonication to form a mixed solution 1; weigh 3 mg of curcumin and dissolve them in 1 mL of ethanol to form a mixed solution 2; weigh 300 mg of hyaluronic acid-TPP and dissolve them in 30 mL of deionized water, and dissolve them by stirring at room temperature to form a mixed solution 3.

[0080] S3. Take 1 mL of mixed solution 2 and slowly drip it into 1 mL of mixed solution 1. Stir and react at room temperature for 30 minutes to form mixed solution 4.

[0081] S4. Take 1 mL of mixed solution 4, slowly drip it into 30 mL of mixed solution 3, stir and react at room temperature for 3 h, and obtain mitochondrial targeted curcumin nanoemulsion.

[0082] Example 4

[0083] A method for preparing a mitochondrial-targeted curcumin nanoemulsion comprises the following steps:

[0084] S1. Preparation of hyaluronic acid-TPP: 100 mg of triphenylphosphine, 2 g of hyaluronic acid, 400 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 100 mg of 4-dimethylaminopyridine were dissolved in 100 ml of water, and an esterification reaction was carried out at room temperature for 15 h. Hyaluronic acid-TPP was obtained by purification by dialysis.

[0085] S2. Weigh 5 mg of zein and 5 mg of lecithin and dissolve them in 1 mL of 80% ethanol, and dissolve them by ultrasonication to form a mixed solution 1; weigh 10 mg of curcumin and dissolve them in 1 mL of ethanol to form a mixed solution 2; weigh 150 mg of hyaluronic acid-TPP and dissolve them in 30 mL of deionized water, and dissolve them by stirring at room temperature to form a mixed solution 3.

[0086] S3. Take 1 mL of mixed solution 2 and slowly drip it into 1 mL of mixed solution 1. Stir and react at room temperature for 30 minutes to form mixed solution 4.

[0087] S4. Take 1 mL of mixed solution 4, slowly drip it into 30 mL of mixed solution 3, and stir the mixture at 30° C. for 1 h to obtain mitochondrial targeted curcumin nanoemulsion.

[0088] Example 5

[0089] A method for preparing a mitochondrial-targeted curcumin nanoemulsion comprises the following steps:

[0090] S1. Preparation of hyaluronic acid-TPP: 100 mg of triphenylphosphine, 2 g of hyaluronic acid, 400 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 100 mg of 4-dimethylaminopyridine were dissolved in 100 ml of water, and esterification reaction was carried out at room temperature for 15 to 24 h. Hyaluronic acid-TPP was obtained by purification by dialysis.

[0091] S2. Weigh 5 mg of zein and 5 mg of lecithin and dissolve them in 1 mL of 80% ethanol, and dissolve them by ultrasonication to form a mixed solution 1; weigh 15 mg of curcumin and dissolve them in 1 mL of ethanol to form a mixed solution 2; weigh 210 mg of hyaluronic acid-TPP and dissolve them in 30 mL of deionized water, and dissolve them by stirring at room temperature to form a mixed solution 3.

[0092] S3. Take 1 mL of mixed solution 2 and slowly drip it into 1 mL of mixed solution 1. Stir and react at room temperature for 30 minutes to form mixed solution 4.

[0093] S4. Take 1 mL of mixed solution 4, slowly drip it into 30 mL of mixed solution 3, and stir the mixture at 45° C. for 1 h to obtain mitochondrial targeted curcumin nanoemulsion.

[0094] The morphology and structure of the mitochondrial targeted curcumin nanoemulsion prepared in Examples 1 to 5 were tested, and the results were as follows:

[0095] Figure 1 This is a microscopic morphology of the mitochondrial targeted curcumin nanoemulsion prepared in Example 1 of the present invention. Figure 1 As shown, the curcumin nanoemulsion is a uniform nanoemulsion.

[0096] Figure 2 This is the particle size distribution diagram of the mitochondrial targeted curcumin nanoemulsion prepared in Example 2 of the present invention. Figure 2 As shown, the particle size of the curcumin nanoemulsion is between 100nm and 700nm.

[0097] The effects of the mitochondrial targeted curcumin nanoemulsions prepared in Examples 1 to 5 are basically the same. Taking Example 1 as an example, the efficacy of the mitochondrial targeted curcumin nanoemulsion was evaluated.

[0098] 1. Conduct mitochondrial targeted testing based on human skin fibroblasts, including the following steps:

[0099] Step 1. Cell recovery and cell passage: Take out HFB cells with a passage number of P6 from the liquid nitrogen tank, recover and inoculate them into culture bottles, and culture them in a 37°C, 5% CO2 incubator. When the cells grow to 70% to 80%, digest and passage them. The digested cells are resuspended in culture medium and counted for later use.

[0100] Step 2: Cell seeding: Add cells to a 12-well plate and plate at 4×10 4 The cells were inoculated into a 12-well plate at a volume of 1 mL per well and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0101] Step 3, experimental grouping: when the cell plating rate reaches 70% to 80%, take out the culture plate and set up the control group and sample group respectively.

[0102] Step 4: Prepare the sample working solution according to the experimental design, as shown in Table 1.

[0103] Table 1 Experimental design of mitochondrial targeting test in human skin fibroblasts

[0104]

[0105] Among them, the detection method is: after the cells are cultured in an incubator for 16 hours, the supernatant is discarded, 500 μL PBS is added to each well for washing, and mitochondrial staining solution is prepared in advance. The mitochondrial staining solution is prepared by mitochondrial green fluorescent probe and serum-free culture medium at a ratio of 1:8000. After discarding PBS, 500 μL mitochondrial staining solution (Mito-Tracker Green, Rhodamine B or Merge) is added to each well, and incubated in a light-proof incubator at 37°C and 5% CO2 for 30 minutes, and then the supernatant is discarded and 500 μL PBS is added to each well for washing 3 times.

[0106] The method of taking pictures is: take out the slide, prepare the sealing solution in advance, use the sealing solution prepared by anti-fluorescence quenching solution and PBS in a ratio of 1:9, take pictures after sealing, take pictures under a 40x fluorescence inverted microscope, and record the two-channel fluorescence photos of each group of cells.

[0107] Fluorescence intensity calculation: Pearson's correlation coefficient of each photo was calculated for each group of samples, and SD value was calculated. Statistical analysis was performed using t-test method to calculate P value.

[0108] Figure 3 This is a microscopic observation result of the targeted fluorescence image of the mitochondrial targeted curcumin nanoemulsion prepared in Example 1 of the present invention.

[0109] Table 2 Fluorescence intensity test results of the mitochondrial targeted curcumin nanoemulsion prepared in Example 1

[0110] Group Pearson's correlation coefficient SD P-value Improvement rate (%) Control group 0.583 0.031 / / Sample set 0.666 0.032 0.0011** 14.14％

[0111] Figure 3 This is a microscopic observation result of the targeted fluorescence image of the mitochondrial targeted curcumin nanoemulsion prepared in Example 1 of the present invention. Figure 4 The bar graph is a result of the targeted test of the mitochondrial curcumin nanoemulsion prepared in Example 1 of the present invention. When the t-test method is used for statistical analysis, * indicates the significance analysis between the "control group" and the "sample group", * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0112] like Figure 3-4 As shown in Table 2, the Pearson's correlation coefficient was between 0.5 and 1.0, indicating that there was co-localization. Both the control group and the sample group had co-localization, and the co-localization effect of the sample group was better than that of the curcumin group, with an improvement rate of 14.14%.

[0113] 2. Antioxidant testing of mitochondrial targeted nanoemulsion based on UVB-induced human skin fibroblasts, including the following steps:

[0114] Step 1, cell recovery and subculture: Take out HFB cells with a passage number of P6 from the liquid nitrogen tank, recover and inoculate them into culture bottles, and culture them in a 37°C, 5% CO2 incubator. When the cells grow to 70% to 80%, digest and subculture them. The digested cells are resuspended in culture medium and counted for later use.

[0115] Step 2, cell inoculation: 5000 cells / well were inoculated into a 96-well plate, incubated overnight in an incubator (37° C., 5% CO 2 ), and the inoculation volume per well was 200 μL.

[0116] Step 3, experimental grouping: The experiment set up a control group, a model group, a curcumin group and a sample group. Each experimental group was set up with 3 replicate wells.

[0117] Step 4, liquid preparation: Prepare the sample working solution according to the experimental design, as shown in Table 3.

[0118] Table 3 Experimental design of UVB-induced antioxidant detection of human skin fibroblasts

[0119]

[0120] Among them, the detection method is: after the cells are cultured in an incubator for 24 hours, the supernatant is discarded, 100 μL PBS is added to each well for washing, and MTT treatment solution is prepared in advance. The MTT treatment solution is prepared by 10% MTT stock solution + 90% culture medium (10% FBS). After discarding PBS, 50 μL MTT treatment solution is added to each well, and incubated in a light-proof incubator at 37°C and 5% CO2 for 2 hours. After that, the supernatant is discarded and 100 μL isopropanol solution is added to each well. The plate is shaken for 30 minutes, and the OD value is read at 570 nm using an enzyme reader.

[0121] Cell viability calculation: survival rate (%) = 100*OD 570e / OD b , O.D. 570e OD is the average optical density of 100% sample extract; b is the average value of blank optical density. Calculate the relative cell viability value and SD value of each group, and use t-test method to perform statistical analysis on P value. Figure 5 And as shown in Table 4.

[0122] Table 4 Relative cell viability of the mitochondrial targeted curcumin nanoemulsion prepared in Example 1

[0123] Group Relative Vitality SD P-value Improvement rate (%) Control group 100.00％ 1.44％ / / Model Group 54.18％ 0.90％ 0.0000*** / Curcumin group 56.27％ 0.86％ 0.4730 / Sample set 81.08％ 2.13％ 0.0027## 49.63％

[0124] Figure 5 This is a bar graph of the cell viability test results of the mitochondrial curcumin nanoemulsion prepared in Example 1 of the present invention. When the t-test method is used for statistical analysis, * indicates the significance analysis between the "model group" and the "control group", * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. # indicates the significance analysis between the "sample group" and the "model group", # indicates P<0.05, ## indicates P<0.01, and ### indicates P<0.001. Figure 5 As shown in Table 4, compared with the control group, the cell viability of the model group was significantly reduced, indicating that the model was successfully established; compared with the model group, the cell viability of the sample group was significantly increased, with an increase rate of 49.63%.

[0125] 3. Detection of reactive oxygen content in human skin fibroblasts induced by UVB based on mitochondria-targeted nanoemulsion, including the following steps:

[0126] Step 1, cell recovery and subculture: Take out HFB cells with a passage number of P6 from the liquid nitrogen tank, recover and inoculate them into culture bottles, and culture them in a 37°C, 5% CO2 incubator. When the cells grow to 70% to 80%, digest and subculture them. The digested cells are resuspended in culture medium and counted for later use.

[0127] Step 2, cell seeding: 3×10 4The cells were inoculated into a 24-well plate at a volume of 0.5 mL per well and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0128] Step 3, experimental grouping: when the cell plating rate reaches 70% to 80%, take out the culture plate and set up the control group, model group, curcumin group and sample group respectively.

[0129] Step 4, liquid preparation: Prepare the sample working solution according to the experimental design, as shown in Table 5.

[0130] Table 5 Experimental design for detecting reactive oxygen species in human skin fibroblasts induced by UVB

[0131]

[0132] The detection method is as follows: perform ROS staining, culture the cells in an incubator for 24 hours, discard the supernatant, add 1 mL of PBS to each well for washing, and then perform staining according to the instructions of the ROS kit. Take photos: After staining, discard the supernatant, add 1 mL of PBS to each well, take photos under a 10x fluorescence inverted microscope, and record the fluorescence photos of each group of ROS.

[0133] Calculation of ROS fluorescence intensity: The average fluorescence intensity of each photo of each group of samples was counted, and the SD value was calculated. The t-test method was used for statistical analysis and the P value was calculated. Figure 6-Figure 7 and as shown in Table 6.

[0134] Table 6 Average fluorescence intensity of ROS of mitochondrial targeted curcumin nanoemulsion prepared in Example 1

[0135] Group Mean fluorescence intensity of ROS SD P-value Decline rate (%) Control group 3.760 0.936 / / Model Group 15.245 2.831 0.0022** / Curcumin group 8.555 3.935 0.0363# 43.88％ Sample set 6.100 1.035 0.0044## 59.99％

[0136] Figure 6 This is a microscopic observation result of ROS fluorescence image of mitochondria-targeted curcumin nanoemulsion prepared in Example 1 of the present invention. Figure 7 The bar graph of the ROS content test results of the mitochondrial curcumin nanoemulsion prepared in Example 1. Wherein, when the t-test method is used for statistical analysis, * represents the significance analysis between the "model group" and the "control group", * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. # represents the significance analysis between the "curcumin group", "sample group" and "model group", # represents P < 0.05, ## represents P < 0.01, and ### represents P < 0.001.

[0137] like Figure 6-7As shown in Table 6, compared with the control group, the mean fluorescence intensity of ROS in the model group was significantly increased, indicating that the model was successfully established; compared with the model group, the mean fluorescence intensity of ROS in the curcumin group and the sample group decreased to varying degrees, with decrease rates of 43.88% and 59.99%, respectively.

[0138] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the attached claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0139] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a mitochondrial-targeted curcumin nanoemulsion, characterized in that: The following steps are involved: Dissolving triphenylphosphine and a carrier in a solvent, and under the action of a condensing agent and / or a catalyst, the triphenylphosphine and the carrier undergo an amidation reaction or an esterification reaction to obtain a carrier-TPP, wherein the carrier-TPP is zein-TPP or hyaluronic acid-TPP; Zein-TPP or zein is used as the core, curcumin is embedded and loaded, and then hyaluronic acid or hyaluronic acid-TPP is used as the shell, and the core and shell are cross-linked to obtain mitochondrial curcumin nanoemulsion; when zein-TPP is the core, hyaluronic acid is used as the shell, and when zein is the core, hyaluronic acid-TPP is used as the shell.

2. The method for preparing the mitochondria-targeted curcumin nanoemulsion according to claim 1, characterized in that: When the carrier-TPP is zein-TPP, the preparation method comprises the following steps: Dissolving triphenylphosphine and zein in a solvent, and causing zein and triphenylphosphine to undergo amidation reaction under the action of a condensing agent to obtain zein-TPP; The mass ratio of zein, triphenylphosphine and condensation agent is 6-22:1:2-13, the second solvent is dimethyl sulfoxide; the condensation agent is a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 1.5-10:1, the temperature of the amidation reaction is 20°C-30°C, and the reaction time is 15h-24h.

3. The method for preparing the mitochondria-targeted curcumin nanoemulsion according to claim 1, characterized in that: When the carrier-TPP is hyaluronic acid-TPP, the preparation method comprises the following steps: Dissolving triphenylphosphine and hyaluronic acid in a solvent, and causing esterification reaction between hyaluronic acid and triphenylphosphine under the action of a condensing agent and a catalyst to obtain hyaluronic acid-TPP; Among them, the mass ratio of hyaluronic acid, triphenylphosphine, condensation agent and catalyst is 18-22:1-3:1-9:0.3-3; the second solvent is water; the condensation agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, the catalyst is 4-dimethylaminopyridine, the temperature of the amidation reaction is 20°C-30°C, and the reaction time is 15h-24h.

4. The method for preparing the mitochondria-targeted curcumin nanoemulsion according to claim 2, characterized in that: When the carrier-TPP is zein-TPP, the preparation method of the mitochondrial-targeted curcumin nanoemulsion comprises the following steps: dissolving zein-TPP and cholesterol or lecithin in a solvent to form a mixed solution A; Curcumin solution is added to mixed solution A to embed curcumin into carrier-TPP to obtain mixed solution B, and then hyaluronic acid solution is added to mixed solution B. Zein-TPP is used as the core and hyaluronic acid is used as the shell. Hyaluronic acid and zein-TPP are cross-linked to obtain mitochondrial curcumin nanoemulsion.

5. The method for preparing the mitochondria-targeted curcumin nanoemulsion according to claim 4, characterized in that: The mass ratio of cholesterol or lecithin to zein-TPP is 1:1-2, the concentration of zein-TPP in the mixed solution A is 5 mg / ml-20 mg / ml, the mass ratio of zein-TPP to curcumin in the curcumin solution is 10:3-30, and the concentration of the curcumin solution is 3 mg / ml-12 mg / ml; the volume ratio of the mixed solution B to the hyaluronic acid solution is 2:30, and the concentration of the hyaluronic acid solution is 5 mg / ml-10 mg / ml.

6. The method for preparing the mitochondria-targeted curcumin nanoemulsion according to claim 3, characterized in that: When the carrier-TPP is hyaluronic acid-TPP, the preparation method of the mitochondrial targeted curcumin nanoemulsion comprises the following steps: dissolving zein and cholesterol or lecithin in a solvent to form a mixed solution C; The curcumin solution is added to the mixed solution C, so that the curcumin is embedded and loaded in the zein to form a mixed solution D, and then the hyaluronic acid-TPP solution is added to the mixed solution D, with the zein as the core and the hyaluronic acid-TPP as the shell, and the hyaluronic acid-TPP and the zein are cross-linked to obtain a mitochondrial-targeted curcumin nanoemulsion.

7. The method for preparing the mitochondria-targeted curcumin nanoemulsion according to claim 6, characterized in that: The mass ratio of cholesterol or lecithin to zein is 1:1-2, the concentration of zein in the mixed solution C is 5 mg / ml-20 mg / ml, the mass ratio of zein to curcumin in the curcumin solution is 10:3-30, the concentration of the curcumin solution is 3 mg / ml-12 mg / ml, the volume ratio of the mixed solution D to the hyaluronic acid-TPP solution is 2:1, and the concentration of the hyaluronic acid-TPP solution is 5 mg / ml-10 mg / ml.

8. The method for preparing the mitochondria-targeted curcumin nanoemulsion according to claim 1, characterized in that: The temperature of the cross-linking reaction is 20°C to 60°C, and the time is 0.5h to 3h.

9. A mitochondrial-targeted curcumin nanoemulsion obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the mitochondria-targeted curcumin nanoemulsion according to claim 9 in preparing cosmetics for delaying skin aging.

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