Deep antioxidant EGCG (epigallocatechin gallate) nano composition as well as preparation method and application thereof

Abstract

The invention relates to a deep antioxidant EGCG (epigallocatechin gallate) nano composition as well as a preparation method and application thereof, and belongs to the technical field of nano carriers. The deep antioxidant EGCG nano composition is prepared from the following raw materials: a carrier, a stabilizer, an active substance, water and polyhydric alcohols, the active substance is EGCG; the mass percent of the EGCG in the preparation raw materials is 0.5-5%; the stabilizer is selected from phytosphingosine and derivatives thereof; the carrier is an amphiphilic polymer, the general formula is Xm-Yn, Xm is polyglycolide, and Yn is poly (2-methacryloyloxyethyl phosphorylcholine). The nano composition is prepared by self-assembling and entrapment through a specific preparation method and then removing polyhydric alcohols in the raw materials. The nano composition provided by the invention is high in encapsulation efficiency, good in stability and good in transdermal absorption, and can play an excellent deep antioxidant effect on skin when being used in cosmetics.

Description

Technical Field

[0001] The present invention relates to the field of nanocarrier technology, and in particular to a deep antioxidant EGCG nanocomposition and a preparation method and use thereof. Background Art

[0002] Epigallocatechin gallate (EGCG) is an ingredient extracted from Chinese green tea. It is the primary active ingredient in green tea and the most abundant catechin component, accounting for 9-13% of green tea's gross weight. EGCG has a unique stereochemical structure and possesses very strong antioxidant activity. Its molecular structure contains eight phenolic hydroxyl groups and two chroman rings. It has a strong ability to scavenge oxygen free radicals, protecting cells and DNA from damage. It effectively neutralizes ROS / RNS through electron and proton transfer mechanisms and can chelate metal ions to block oxidative chain reactions.

[0003] EGCG can be used as an antioxidant active ingredient to resist skin oxidation and aging. However, there are three major application bottlenecks in the actual application of EGCG in the cosmetics field: ① In terms of stability, because EGCG is rich in phenolic hydroxyl groups in its molecular structure, phenolic hydroxyl groups are easily oxidized to form red quinone derivatives under light (especially UVB), high temperature (>40°C) and pH>6, and the antioxidant effect is correspondingly weakened, resulting in browning of the product and an activity decay rate of up to 80%; ② In terms of transdermal absorption, the high water solubility of EGCG conflicts with the stratum corneum lipid barrier (composed of ceramides, cholesterol, etc., which is fat-soluble). EGCG often only stays on the outermost part of the stratum corneum and cannot effectively penetrate the stratum corneum of the skin to better exert its efficacy. The transdermal rate of conventional formulas is very low, and nanoliposomes or microemulsion carriers are needed to improve delivery efficiency; ③ In terms of bioavailability, metabolic enzymes on the skin surface can quickly degrade EGCG.

[0004] In order to solve the problems existing in the existing technology when adding EGCG to cosmetics and broaden the use of EGCG in cosmetics, it is urgent to develop a strategy that can improve the stability and transdermal absorption effect of EGCG and realize the deep antioxidant function of EGCG on the skin. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provide a deep antioxidant EGCG nanocomposition, its preparation method, and its use. The nanocomposition of the present invention has a high encapsulation efficiency of EGCG, good storage stability, high transdermal absorption and cell affinity, and excellent deep antioxidant efficacy.

[0006] To achieve the above object, the technical solution adopted by the present invention is: In a first aspect, the present invention provides a deep antioxidant EGCG nanocomposition, wherein the raw materials for preparing the deep antioxidant EGCG nanocomposition include a carrier, a stabilizer, an active substance, water, and a polyol; The active substance is EGCG; the mass percentage of EGCG in the raw materials for preparing the deep antioxidant EGCG nanocomposition is 0.5-5%; The stabilizer is at least one of phytosphingosine and its derivatives; The carrier is an amphiphilic polymer, and the general formula of the amphiphilic polymer is X m -Y n , the X m is polyglycolide, said Y n It is poly 2-methacryloyloxyethyl phosphorylcholine.

[0007] The present invention uses an amphiphilic polymer with specific hydrophobic and hydrophilic segments and an antioxidant active ingredient ECGG to achieve self-assembly in a specific type of stabilizer during the preparation process, thereby obtaining a deep antioxidant EGCG nanocomposition that can significantly improve the transdermal absorption of EGCG, enhance the encapsulation rate and stability, and at the same time has high cell affinity and deep antioxidant efficacy.

[0008] In the amphiphilic polymer used, the hydrophobic segment is composed of polyglycolide (PGA): the strong intermolecular force of PGA can enhance the physical stability of the formed nanocomposition, and its appropriate hydrophobicity can promote the compatibility of the nanocomposition with the skin lipid layer, thereby enhancing the transdermal efficiency of EGCG through mechanisms such as stratum corneum penetration; at the same time, the hydrophobic interaction between PGA and the phospholipid bilayer of the skin cell membrane can enhance its fusion efficiency with the cell membrane and improve the intracellular delivery effect; in addition, PGA is a biodegradable material, and its degradation rate matches the cell metabolic cycle, which can reduce irritation to cells, has high cell affinity, and good biocompatibility.

[0009] The hydrophilic segment of the amphiphilic polymer is composed of poly (2-methacryloyloxyethyl) phosphorylcholine (PMPC), a zwitterionic polymer with both positive and negatively charged groups within the molecule. This can reduce the aggregation of the nanocomposite through electrostatic shielding, improving its stability. PMPC also enhances its compatibility with the skin's lipid layer (primarily composed of ceramide and cholesterol), strengthening the intercellular lipid pathways in the stratum corneum and promoting the penetration of the nanocomposite. By integrating the hydrophilic and hydrophobic segments, the present invention uses the amphiphilic PMPC hydrophilic segment to connect and encapsulate EGCG, stabilizing the catechol groups of EGCG. This increases its antioxidant activity retention and reduces its contact with external moisture, oxygen, and metal ions, comprehensively improving the antioxidant efficacy of EGCG when used in cosmetics.

[0010] Specific stabilizers, phytosphingosine and its derivatives, are selected. The lipid portion of the phytosphingosine can combine with the hydrophobic segment of the amphiphilic polymer through hydrophobic interaction to form more stable micelles and enhance stability. Phytosphingosine substances can also reduce the metabolic loss of the nanocomposition in the epidermis by enhancing the skin barrier function. At the same time, they can repair the skin barrier, increase the fluidity of stratum corneum lipids, and reduce the penetration resistance of the nanocomposition. Through structural interaction and enhanced membrane affinity with amphiphilic polymers, they can significantly improve the transdermal efficiency and stability of the nanocomposition and enhance the deep antioxidant effect.

[0011] The present invention uses an amphiphilic polymer with PGA as the hydrophobic segment and PMPC as the hydrophilic segment to encapsulate a specific amount of EGCG. The addition of a specific stabilizer further improves performance, effectively enhancing the stability, transdermal permeability, cell affinity, and antioxidant properties of EGCG. Compared to free EGCG or liposomes and nanoemulsions commonly used in the prior art, the nanocomposite of the present invention exhibits higher stability, better transdermal permeability, and superior antioxidant efficacy. It can be delivered deep into skin cells, effectively regulating antioxidant gene expression and achieving comprehensive, deeper antioxidant efficacy.

[0012] Preferably, the preparation method of the amphiphilic polymer comprises the following steps: S1. Mix the initiator, glycolide and catalyst, and react at 80-150° C. for 6-24 hours to obtain polyglycolide; S2, adding the polyglycolide and the bromination reagent to a solvent and mixing them evenly, and reacting them at 0-25° C. for 12-36 hours to obtain brominated polyglycolide; S3, adding the brominated polyglycolide, 2-methacryloyloxyethyl phosphorylcholine, catalyst and catalyst ligand to the solvent, mixing, and reacting at 60-100° C. for 24-72 hours to obtain the amphiphilic polymer; the reactions in S1, S2 and S3 are all carried out under an inert atmosphere.

[0013] Preferably, in step S1, the catalyst is at least one of stannous octoate, stannous isooctanoate, stannous tartrate, and stannous stearate, and the initiator is a C1-C6 monohydric alcohol; Preferably, in step S1, the molar ratio of the initiator, glycolide and catalyst is initiator:glycolide:catalyst=1:(20-40):(2-4); Preferably, in step S2, the bromination reagent is at least one of 2-bromoisobutyryl bromide, tert-butyl 2-bromoisobutyrate, and ethyl 2-bromoisovalerate; Preferably, in step S2, the molar ratio of polyglycolide to the bromination reagent is 1:(2-8); Preferably, in step S3, the catalyst is at least one of CuBr, CuCl, and CuI, and the catalyst ligand is an amine or ammonium catalyst ligand; Preferably, in step S3, the molar ratio of brominated polyglycolide, 2-methacryloyloxyethyl phosphorylcholine, catalyst, and catalyst ligand is brominated polyglycolide: 2-methacryloyloxyethyl phosphorylcholine: catalyst: catalyst ligand = 1: (80-160): (0.8-1.2): (8-16).

[0014] More preferably, the initiator is at least one of methanol, ethanol, n-propanol, 2-propanol, n-butanol, 2-butanol, n-pentanol, 2-pentanol, 3-pentanol, n-hexanol, 2-hexanol, and 3-hexanol.

[0015] Further preferably, the catalyst ligand is at least one of N,N-diisopropylethylamine, ethanolamine, N,N,N',N',N"-pentamethyldiethylenetriamine, and tetrabutylammonium bromide.

[0016] Further preferably, the solvent is a solvent commonly used in the art, and can be selected from at least one of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, dichloromethane, tetrahydrofuran, acetone, chloroform, acetonitrile, and ethyl acetate.

[0017] Further preferably, the inert gas is an inert gas commonly used in the art, and can be selected from one or more of nitrogen, argon, and helium, with argon being preferred.

[0018] Further preferably, after the reaction of the amphiphilic polymer is completed, a purification process is further included. The purification process specifically comprises: slowly adding the amphiphilic polymer to a precipitant, subjecting the amphiphilic polymer to a precipitation purification process, filtering, washing the solid with the precipitant multiple times, and drying at 25-80°C for 12-72 hours to obtain a refined amphiphilic polymer; the precipitant comprises at least one of methanol, ethanol, n-hexane, and petroleum ether; and the precipitation purification process is performed at a temperature of -10-10°C.

[0019] In the preparation step of the amphiphilic polymer, the hydrophobic segment polyglycolide (PGA) of the amphiphilic polymer is obtained from the polymer monomer glycolide through ring-opening polymerization (ROP); the hydrophilic block poly 2-methacryloyloxyethyl phosphorylcholine (PMPC) of the amphiphilic polymer is obtained from the polymer monomer 2-methacryloyloxyethyl phosphorylcholine through atom transfer radical polymerization (ATRP); finally, the hydrophobic segment and the hydrophilic segment are connected by reaction under the action of an initiator molecule to obtain the amphiphilic polymer.

[0020] Preferably, the degree of polymerization of the polyglycolide is m=40-80; Preferably, the degree of polymerization of the poly (2-methacryloyloxyethyl) phosphorylcholine is n=80-160.

[0021] Preferably, the stabilizer is at least one of phytosphingosine, tetraacetyl phytosphingosine, and phytosphingosine salicylate; Preferably, the polyol is C2-C 10 Polyols.

[0022] More preferably, the polyol is at least one of 1,2-propylene glycol, 1,3-propylene glycol, 1,3-butylene glycol, 1,2-butylene glycol, 1,4-butylene glycol, dipropylene glycol, 1,2-hexanediol, 1,6-hexanediol, ethoxydiglycol, 1,8-octanediol, and 1,10-decanediol.

[0023] More preferably, the stabilizer is phytosphingosine.

[0024] Different stabilizer selections have an impact on the storage stability, transdermal permeability and cell affinity of the nanocomposition prepared by the present invention. The use of a preferred stabilizer can significantly improve the comprehensive performance of the nanocomposition.

[0025] Preferably, in the preparation raw materials, the mass percentage of the amphiphilic polymer is 0.5-5%, the mass percentage of EGCG is 0.5-5%, the mass percentage of the stabilizer is 0.05-1%, the mass percentage of the polyol is 5-30%, and the balance is water.

[0026] Preferably, the mass percentage of the amphiphilic polymer in the preparation raw material is within the range of one or any two of 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, and 5.0%.

[0027] Preferably, the mass percentage of EGCG in the preparation raw materials is within the range of one or any two of 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, and 5.0%.

[0028] Further preferably, the polymerization degree m of the polyglycolide is 50-70, and the polymerization degree n of the poly-2-methacryloyloxyethyl phosphorylcholine is 100-140; Preferably, in the preparation raw materials, the mass percentage of the amphiphilic polymer is 2-4%, the mass percentage of EGCG is 2-4%, the mass percentage of the stabilizer is 0.2-0.5%, the mass percentage of the polyol is 20-30%, and the balance is water.

[0029] The ratio of the amphiphilic polymer and EGCG components during the preparation of the nanocomposite, as well as the degree of polymerization of the hydrophobic and hydrophilic segments in the amphiphilic polymer, will ultimately affect the storage performance, transdermal absorption performance, cell affinity and antioxidant efficacy of the nanocomposite during use. Under the specified preferred degree of polymerization of the hydrophobic and hydrophilic segments, the nanocomposite has excellent cell affinity, which is more conducive to its efficacy at the cellular level.

[0030] In a second aspect, the present invention provides a method for preparing the above-mentioned deep antioxidant EGCG nanocomposition, comprising the following steps: (1) heating and mixing the amphiphilic polymer, stabilizer, and EGCG in a polyol to obtain an alcohol phase solution; (2) adding the alcohol phase solution dropwise to water while subjecting it to high shear treatment to obtain a mixed solution; (3) removing the polyol from the mixed solution and then homogenizing it to obtain the deep antioxidant EGCG nanocomposite.

[0031] During the preparation process, the alcohol phase solution is subjected to high shear treatment to initially prepare self-assembled nanomaterials. At this time, the polyol needs to be removed, and the remaining nanocomposite forms a more uniform dispersion system through hydrogen bonds and van der Waals forces, thereby enhancing the interfacial bonding strength of the composite material. At the same time, after removing the polyol, the high specific surface area and surface activity of the formed nanocomposite can be more fully utilized.

[0032] Preferably, in step (1), the temperature for heating and mixing is 35-80°C; Preferably, in step (2), the rate of adding the alcohol phase solution is 1-10 mL / min, the speed of the high shear treatment is 6000-12000 rpm, and the high shear treatment is performed until the alcohol phase solution is added dropwise; Preferably, in step (3), dialysis is used to remove the polyol, and the pore size of the dialysis bag is 300-1000Da; Preferably, the pressure of the homogenization treatment in step (3) is 50-120 MPa, and the number of homogenization cycles is 2-8 times.

[0033] The present invention uses a dialysis bag with a specific pore size for dialysis operation, so that the molecular weight cutoff of the dialysis bag is greater than the molecular weight of the polyol and the pore size is much smaller than the particle size of the nanocomposite, thereby successfully removing the polyol in the solution and retaining the prepared deep antioxidant EGCG nanocomposite.

[0034] As a preferred embodiment of the present invention, in step (1), the stirring speed for heating and mixing is 200-500 rpm; and the stirring time is 1-6 h.

[0035] As a preferred embodiment of the present invention, in step (2), after the dropwise addition is completed, stirring is carried out at 200-500 rpm for 1-6 hours.

[0036] As a preferred embodiment of the present invention, in step (3), the dialysis treatment time is 12-72 hours.

[0037] As a preferred embodiment of the present invention, the stirring method is mechanical stirring or magnetic stirring.

[0038] The deep antioxidant EGCG nanocomposition prepared by the above preparation method can achieve an encapsulation rate of EGCG of more than 93% and good stability; it also has good transdermal absorption, a high cumulative permeation per unit area, and can take into account better cell affinity, and the average survival rate of cells treated for a long time at a concentration of 1% is higher than 95%; this deep antioxidant nanocomposition comprehensively improves the antioxidant performance, can upregulate the NQO1, CAT, Nrf2, SOD1, and SOD2 genes in cells, promote the activation of antioxidant defense mechanisms at the cellular gene level, and achieve a deep antioxidant effect on skin cells.

[0039] In a third aspect, the present invention provides use of the deep antioxidant EGCG nanocomposition in antioxidant cosmetics.

[0040] Preferably, the use is to add the deep antioxidant EGCG nanocomposition into antioxidant cosmetics; the addition amount of the deep antioxidant EGCG nanocomposition is 0.1-5% by mass.

[0041] Compared with the prior art, the present invention has the following beneficial effects: By optimizing amphiphilic polymers, selecting specific hydrophobic and hydrophilic segments, and using phytosphingosine and its derivatives as stabilizers, the nanocomposite, which is then self-assembled with the antioxidant active ingredient ECGG under specific preparation steps, provides a deep antioxidant EGCG nanocomposite that significantly improves transdermal absorption of EGCG, increases encapsulation efficiency and stability, and simultaneously exhibits high cell affinity and deep antioxidant efficacy. This nanocomposite addresses the existing issues of EGCG's poor stability, poor transdermal absorption, and inability to penetrate skin cells to exert its deep antioxidant effects, thus providing a highly valuable application. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is a synthetic route for the amphiphilic polymers in Examples 1-5; Figure 2 is the GPC curve diagram of the amphiphilic polymer in Examples 1-5; Figure 3 The results of the expression of the antioxidant NQO1 gene in HaCaT cells repairing oxidative damage; Figure 4 The results of the expression of antioxidant CAT gene in HaCaT cells repairing oxidative damage; Figure 5 The results of the expression of the antioxidant Nrf2 gene in HaCaT cells repairing oxidative damage; Figure 6 The results of the expression of the antioxidant SOD1 gene in HaCaT cells repairing oxidative damage; Figure 7 The results of the expression of the antioxidant SOD2 gene in HaCaT cells repairing oxidative damage. DETAILED DESCRIPTION

[0043] To better illustrate the objectives, technical solutions, and advantages of the present invention, the present invention will be further described below with reference to specific examples. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0044] In the examples and comparative examples of the present invention, EGCG, phytosphingosine, tetraacetyl phytosphingosine and phytosphingosine salicylate used were purchased from Guangzhou Xiyuan Biotechnology Co., Ltd. The synthetic route of the amphiphilic polymer in the following embodiment is as follows Figure 1 ; The molecular weight of the amphiphilic polymers in the following examples was determined using a gel permeation chromatograph (GPC, Waters Corporation, USA; model: Waters 1525 / 2414) using a chromatographically pure THF solution as the mobile phase at a flow rate of 1.0 mL / min. The chromatographic column was calibrated with a monodistributed polystyrene standard and the test temperature was 30°C.

[0045] Example 1 An embodiment of the deep antioxidant EGCG nanocomposition of the present invention, the raw materials for preparing the deep antioxidant EGCG nanocomposition in this embodiment include the following components in percentage by weight: 3% of the amphiphilic polymer carrier PGA 60 -PMPC 120 , 0.3% stabilizer phytosphingosine, 3% active substance EGCG, 20% 1,3-butanediol, and the balance is water.

[0046] The amphiphilic polymer used in this example is PGA 60 -PMPC 120 , its preparation method is as follows (taking the preparation of 1.0 mmol as an example): S1. Ethanol (46.07 mg, 1.0 mmol), glycolide GA (3.48 g, 30.0 mmol), and stannous octoate (1.22 mg, 3.0 mmol) were mixed evenly, purged with argon, and reacted at 130°C for 12 h. The reaction mixture was then added dropwise to an excess of petroleum ether (5°C). After precipitation and purification, the solid product was collected and dried at 50°C for 24 h to obtain polyglycolide PGA. 60 .

[0047] S2, PGA 60 (3.48 g, 1.0 mmol) was dissolved in tetrahydrofuran (100 mL), and argon was passed through. 2-bromoisobutyryl bromide (0.92 g, 4.0 mmol) was added and reacted at 20°C for 24 h. The reaction mixture was then added to excess petroleum ether (5°C). After precipitation and purification, the solid product was collected and dried at 50°C for 24 h to obtain brominated PGA. 60 .

[0048] S3, brominated PGA 60 (3.61 g, 1.0 mmol), 2-methacryloyloxyethyl phosphorylcholine (35.44 g, 120.0 mmol), and N,N,N',N',N"-pentamethyldiethylenetriamine (2.08 g, 12.0 mmol) were dissolved in tetrahydrofuran (100 mL). Under argon protection, catalyst CuBr (168 mg, 1.2 mmol) was added and reacted at 65°C for 48 h. The reaction mixture was then added to excess petroleum ether (5°C). After precipitation and purification, the solid product was collected and dried at 50°C for 24 h to obtain the amphiphilic polymer PGA. 60 -PMPC 120 (GPC curve as Figure 2 , calculated Mn = 37812).

[0049] The preparation method of the deep antioxidant EGCG nanocomposition described in this embodiment is: (1) The prepared amphiphilic polymer PGA 60 -PMPC 120 , stabilizer phytosphingosine and EGCG were added to 1,3-butanediol, heated to 60°C, and stirred at 400 rpm for 3 h to obtain an alcohol phase solution; (2) The alcohol phase solution was added dropwise to deionized water at a dropping rate of 5 mL / min, and high-speed shearing treatment was simultaneously started at a speed of 8000 rpm; after the addition was completed, stirring was continued at a speed of 400 rpm for 3 h to obtain a mixed solution; (3) The mixed solution was transferred to a dialysis bag (with a molecular weight cut-off of 500 Da) and dialyzed in water 10 times the volume of the mixed solution for 48 h to replace the 1,3-butanediol in the mixed solution with deionized water, with the deionized water being replaced every 6 h. After the dialysis, the solution in the dialysis bag was taken out and homogenized by microjet high pressure with the pressure adjusted to 70 MPa for 5 times to obtain the deep antioxidant EGCG nanocomposition.

[0050] Example 2 An embodiment of the deep antioxidant EGCG nanocomposition of the present invention is different from Example 1 only in that the carrier amphiphilic polymer used is PGA 40 -PMPC 120 The preparation method is as follows (taking the preparation of 1.0 mmol as an example): S1. Ethanol (46.07 mg, 1.0 mmol), glycolide GA (2.32 g, 20.0 mmol), and stannous octoate (0.81 mg, 2.0 mmol) are mixed evenly, protected by argon, and reacted at 130°C for 12 h; then the reaction mixture is added dropwise to excess petroleum ether (5°C), and after precipitation and purification, the solid product is collected and dried at 50°C for 24 h to obtain polyglycolide PGA. 40 .

[0051] S2, PGA 40 (2.32 g, 1.0 mmol) was dissolved in tetrahydrofuran (100 mL), and argon was passed through. 2-bromoisobutyryl bromide (0.92 g, 4.0 mmol) was added and reacted at 20°C for 24 h. The reaction mixture was then added to excess petroleum ether (5°C). After precipitation and purification, the solid product was collected and dried at 50°C for 24 h to obtain brominated PGA. 40 .

[0052] S3, brominated PGA 40 (2.45 g, 1.0 mmol), 2-methacryloyloxyethyl phosphorylcholine (35.44 g, 120.0 mmol), and N,N,N',N',N"-pentamethyldiethylenetriamine (2.08 g, 12.0 mmol) were dissolved in tetrahydrofuran (100 mL), and under argon protection, catalyst CuBr (168 mg, 1.2 mmol) was added, and the reaction was carried out at 65 ° C for 48 h; then the reaction mixture was added to excess petroleum ether (5 ° C), and after precipitation and purification, the solid product was collected and dried at 50 ° C for 24 h to obtain the amphiphilic polymer PGA. 40 -PMPC 120 (GPC curve as Figure 2 , calculated Mn = 36008).

[0053] The preparation method of the deep antioxidant EGCG nanocomposition described in this example refers to Example 1.

[0054] Example 3 An embodiment of the deep antioxidant EGCG nanocomposition of the present invention is different from Example 1 only in that the carrier amphiphilic polymer used is PGA 80 -PMPC 120 The preparation method is as follows (taking the preparation of 1.0 mmol as an example): S1. Ethanol (46.07 mg, 1.0 mmol), glycolide GA (4.64 g, 40.0 mmol), and stannous octoate (1.62 mg, 4.0 mmol) are mixed evenly, protected by argon, and reacted at 130°C for 12 h; then the reaction mixture is added dropwise to excess petroleum ether (5°C), and after precipitation and purification, the solid product is collected and dried at 50°C for 24 h to obtain polyglycolide PGA. 80 .

[0055] S2, PGA 80 (4.64 g, 1.0 mmol) was dissolved in tetrahydrofuran (100 mL), and argon was passed through. 2-bromoisobutyryl bromide (0.92 g, 4.0 mmol) was added and reacted at 20°C for 24 h. The reaction mixture was then added to excess petroleum ether (5°C). After precipitation and purification, the solid product was collected and dried at 50°C for 24 h to obtain bromo-PGA. 80 .

[0056] S3, brominated PGA 80 (4.77 g, 1.0 mmol), 2-methacryloyloxyethyl phosphorylcholine (35.44 g, 120.0 mmol), and N,N,N',N',N"-pentamethyldiethylenetriamine (2.08 g, 12.0 mmol) were dissolved in tetrahydrofuran (100 mL), and under argon protection, catalyst CuBr (168 mg, 1.2 mmol) was added, and the reaction was carried out at 65 ° C for 48 h; then the reaction mixture was added to excess petroleum ether (5 ° C), and after precipitation and purification, the solid product was collected and dried at 50 ° C for 24 h to obtain the amphiphilic polymer PGA. 80 -PMPC 120 (GPC curve as Figure 2 , calculated Mn = 38457).

[0057] The preparation method of the deep antioxidant EGCG nanocomposition described in this example refers to Example 1.

[0058] Example 4 An embodiment of the deep antioxidant EGCG nanocomposition of the present invention is different from Example 1 only in that the carrier amphiphilic polymer used is PGA 60 -PMPC 80 The preparation method is as follows (taking the preparation of 1.0 mmol as an example): S1. Ethanol (46.07 mg, 1.0 mmol), glycolide GA (3.48 g, 30.0 mmol), and stannous octoate (1.22 mg, 3.0 mmol) are mixed evenly, protected by argon, and reacted at 130°C for 12 h; then the reaction mixture is added dropwise to excess petroleum ether (5°C), and after precipitation and purification, the solid product is collected and dried at 50°C for 24 h to obtain polyglycolide PGA. 60 .

[0059] S2, PGA 60 (3.48 g, 1.0 mmol) was dissolved in tetrahydrofuran (100 mL), and argon was passed through. 2-bromoisobutyryl bromide (0.92 g, 4.0 mmol) was added and reacted at 20°C for 24 h. The reaction mixture was then added to excess petroleum ether (5°C). After precipitation and purification, the solid product was collected and dried at 50°C for 24 h to obtain brominated PGA. 60 .

[0060] S3, brominated PGA 60 (3.61 g, 1.0 mmol), 2-methacryloyloxyethyl phosphorylcholine (23.63 g, 80.0 mmol), and N,N,N',N',N"-pentamethyldiethylenetriamine (1.39 g, 8.0 mmol) were dissolved in tetrahydrofuran (100 mL). Under argon protection, catalyst CuBr (112 mg, 0.8 mmol) was added and reacted at 65°C for 48 h. The reaction mixture was then added to excess petroleum ether (5°C). After precipitation and purification, the solid product was collected and dried at 50°C for 24 h to obtain the amphiphilic polymer PGA. 60 -PMPC 80 (GPC curve as Figure 2 , calculated Mn = 25984).

[0061] The preparation method of the deep antioxidant EGCG nanocomposition described in this example refers to Example 1.

[0062] Example 5 An embodiment of the deep antioxidant EGCG nanocomposition of the present invention is different from Example 1 only in that the carrier amphiphilic polymer used is PGA 60 -PMPC 160The preparation method is as follows (taking the preparation of 1.0 mmol as an example): S1. Ethanol (46.07 mg, 1.0 mmol), glycolide GA (3.48 g, 30.0 mmol), and stannous octoate (1.22 mg, 3.0 mmol) are mixed evenly, protected by argon, and reacted at 130°C for 12 h; then the reaction mixture is added dropwise to excess petroleum ether (5°C), and after precipitation and purification, the solid product is collected and dried at 50°C for 24 h to obtain polyglycolide PGA. 60 .

[0063] S2, PGA 60 (3.48 g, 1.0 mmol) was dissolved in tetrahydrofuran (100 mL), and argon was passed through. 2-bromoisobutyryl bromide (0.92 g, 4.0 mmol) was added and reacted at 20°C for 24 h. The reaction mixture was then added to excess petroleum ether (5°C). After precipitation and purification, the solid product was collected and dried at 50°C for 24 h to obtain brominated PGA. 60 .

[0064] S3, brominated PGA 60 (3.61 g, 1.0 mmol), 2-methacryloyloxyethyl phosphorylcholine (47.26 g, 160.0 mmol), and N,N,N',N',N"-pentamethyldiethylenetriamine (2.78 g, 16.0 mmol) were dissolved in tetrahydrofuran (100 mL), and under argon protection, catalyst CuBr (224 mg, 1.6 mmol) was added, and the reaction was carried out at 65 ° C for 48 h; then the reaction mixture was added to excess petroleum ether (5 ° C), and after precipitation and purification, the solid product was collected and dried at 50 ° C for 24 h to obtain the amphiphilic polymer PGA. 60 -PMPC 160 (GPC curve as Figure 2 , calculated Mn = 49157).

[0065] The preparation method of the deep antioxidant EGCG nanocomposition described in this example refers to Example 1.

[0066] Example 6 An embodiment of the deep antioxidant EGCG nanocomposition of the present invention is different from Example 1 only in that the stabilizer phytosphingosine is replaced with tetraacetyl phytosphingosine with the same mass content.

[0067] Example 7 An embodiment of the deep antioxidant EGCG nanocomposition of the present invention is different from Example 1 only in that the stabilizer phytosphingosine is replaced by phytosphingosine salicylate with the same mass content.

[0068] Example 8 An embodiment of the deep antioxidant EGCG nanocomposition of the present invention is different from Example 1 only in that the carrier amphiphilic polymer PGA is changed. 60 -PMPC 120 The amount of deionized water was adjusted so that the mass percentage of the other components remained unchanged.

[0069] Example 9 An embodiment of the deep antioxidant EGCG nanocomposition of the present invention is different from Example 1 only in that the carrier amphiphilic polymer PGA is changed. 60 -PMPC 120 The amount of deionized water was adjusted so that the mass percentage of the other components remained unchanged.

[0070] Example 10 An embodiment of the deep antioxidant EGCG nanocomposition of the present invention is different from Example 1 only in that the carrier amphiphilic polymer PGA is changed. 60 -PMPC 120 The amount of deionized water was adjusted so that the mass percentage of the other components remained unchanged.

[0071] Example 11 An embodiment of the deep antioxidant EGCG nanocomposition of the present invention is different from Example 1 only in that the amount of the active substance EGCG is changed so that its mass percentage in the preparation raw materials is 0.5%, and the amount of deionized water is adjusted so that the mass percentages of other components remain unchanged.

[0072] Example 12 An embodiment of the deep antioxidant EGCG nanocomposition of the present invention is different from Example 1 only in that the amount of the active substance EGCG is changed to 1% by mass in the preparation raw materials, and the amount of deionized water is adjusted to keep the mass percentages of other components unchanged.

[0073] Example 13 An embodiment of the deep antioxidant EGCG nanocomposition of the present invention is different from Example 1 only in that the amount of the active substance EGCG is changed so that its mass percentage in the preparation raw materials is 5%, and the amount of deionized water is adjusted so that the mass percentages of other components remain unchanged.

[0074] Comparative Example 1 The only difference between Comparative Example 1 and Example 1 is that the stabilizer phytosphingosine is not added, and the amount of deionized water is adjusted so that the mass percentages of other components remain unchanged.

[0075] Comparative Example 2 The only difference between Comparative Example 2 and Example 1 is that the amount of the active substance EGCG is changed so that its mass percentage in the raw materials is 7%, and the amount of deionized water is adjusted so that the mass percentages of other components remain unchanged.

[0076] Comparative Example 3 The only difference between Comparative Example 3 and Example 1 is that, during the preparation process, the dialysis operation in step (3) is not performed, and the mixed solution obtained in step (2) is directly subjected to microjet high-pressure homogenization, the pressure is adjusted to 70 MPa, and the process is performed 5 times to obtain the deep antioxidant nanocomposition.

[0077] Comparative Example 4 Calculated by weight percentage, 3% soy lecithin, 0.1% cholesterol, and 3% EGCG were dissolved in ethanol with a weight percentage of 30% of the finished product, and stirred at 400 rpm at 60° C. for 3 h to obtain a mixed solution; The resulting mixed solution was subjected to rotary evaporation (40°C, 250 rpm) to remove the ethanol solution; after the ethanol was completely removed, deionized water (93.9% by mass of the finished product) was added, and the mixture was subjected to 600W ultrasonic dispersion for 30 minutes. Finally, microfluidization high-pressure homogenization was performed at a pressure of 70 MPa for 5 times to obtain EGCG-encapsulated liposomes.

[0078] Comparative Example 5 In terms of mass percentage, 3% EGCG and 15% caprylic / capric triglyceride were mixed and heated to 85° C. until the EGCG was completely dissolved, and then 10% butylene glycol was added to obtain an oil phase component; The oil phase was uniformly added dropwise to 72% by weight deionized water at a dropping rate of 3 mL / min. After the addition was complete, stirring was continued at 300 rpm for 15 minutes to obtain a mixed solution. The mixed solution was dispersed by high-speed shearing at 10,000 rpm for 20 minutes, and then homogenized by high-pressure microfluidization at a homogenization pressure of 80 MPa and four times to obtain an EGCG nanoemulsion.

[0079] Comparative Example 6 The carrier amphiphilic polymer PGA used in Comparative Example 6 60 -PMPC 120 The mass percentages of the raw materials used in the preparation are the same as those in Example 1, and the preparation method of the nanocomposite is as follows: (1) The prepared amphiphilic polymer PGA 60 -PMPC 120 , stabilizer phytosphingosine and EGCG were added to 1,3-butanediol and heated to 60°C to obtain an alcohol phase solution; EGCG was added to deionized water and stirred at 400 rpm for 3 h to obtain an aqueous solution; (2) adding the alcohol phase solution to the aqueous phase solution, simultaneously starting high-speed shearing at a speed of 8000 rpm and a dropping rate of 5 mL / min; after the dropping is completed, stirring is continued at a speed of 400 rpm for 3 h to obtain a mixed solution; (3) The dialysis and high-pressure homogenization operations in step (3) are the same as those in Example 1 to obtain a nanocomposite.

[0080] Comparative Example 7 In Comparative Example 7, 3% by mass of EGCG was added to 97% by mass of deionized water, and the mixture was stirred at 300 rpm for 15 minutes until the EGCG was completely dissolved to obtain an EGCG aqueous solution.

[0081] Effect Example 1 In order to explore the encapsulation effect and storage stability of the deep antioxidant EGCG nanocomposition provided by the present invention, the nanocompositions or other products in the above examples and comparative examples were subjected to the following tests: Encapsulation efficiency test: 200 μL of the above-mentioned sample was taken and ultrafiltration centrifuged (9000 rpm, 30 min). 5 μL of the filtrate was taken and the EGCG content in the filtrate was determined by high performance liquid chromatography (HPLC, Shimadzu, Japan) to determine the content of free (unencapsulated) EGCG in the sample.

[0082] Separately, the above-mentioned sample was added to a mixed solution of methanol and deionized water. Ultrasonic demulsification was performed at a ratio of sample: methanol: deionized water = 1:4:5 (v / v) for 30 min. After filtration through a 0.45 μm organic filter membrane, 5 μL of the sample solution was measured by high-performance liquid chromatography (HPLC, Shimadzu, Japan) to determine the EGCG content. The total EGCG content in the EGCG preparation was obtained. The encapsulation efficiency (EE) of the sample was calculated according to the following formula: Wherein, C1 represents the content of free (unencapsulated) EGCG in the sample; C0 represents the total content of EGCG in the sample after ultrasonic demulsification of a mixed solution of methanol and deionized water.

[0083] Storage stability test: The samples in the examples and comparative examples were stored at room temperature (25°C) for one month, two months, and three months, and the encapsulation efficiency and EGCG content of the samples were measured to test the storage stability of the samples. The results of the encapsulation efficiency and storage stability are shown in Table 1.

[0084] Table 1 Encapsulation efficiency and storage stability results of Examples and Comparative Examples From Table 1 we can see that: Comparing Examples 1-5, it can be seen that the different degrees of polymerization (DPs) of the hydrophobic block PGA and the hydrophilic block PMPC in the amphiphilic polymers used to prepare the nanocomposites affect the storage stability of the resulting nanocomposites. In Examples 1-3, the DPs of the hydrophobic block PGA were 60, 40, and 80, respectively. As the DP of PGA increased, the changes in the EGCG content and encapsulation efficiency in the nanocomposites after three months of room temperature storage initially decreased and then increased. In Examples 1, 4, and 5, the DPs of the hydrophilic block PMPC were 120, 80, and 160, respectively. Similarly, as the DP of PMPC increased, the changes in EGCG content and encapsulation efficiency after three months of room temperature storage initially decreased and then increased. In Example 1, when the DPs of PGA were 60 and PMPC were 120, the changes in EGCG content and encapsulation efficiency after three months of room temperature storage were minimal, at -10.96% and -10.17%, respectively, demonstrating optimal storage stability.

[0085] Comparison of Examples 1, 6, 7 and Comparative Example 1 shows that the addition of stabilizers and their types also affect storage stability. In Example 1, when phytosphingosine is selected as the stabilizer to interact with the other components, the storage stability is optimal.

[0086] By comparing Examples 1, 8-13 and Comparative Example 2, it can be seen that the different amounts of active ingredients EGCG and amphiphilic polymers affect the storage stability of the product. It can be seen that as the amount of amphiphilic polymer increases from 0.5-5%, or the amount of EGCG increases from 0.5-7%, the change rate of EGCG content and encapsulation efficiency of the deep antioxidant nanocomposition after storage at room temperature for 3 months first decreases and then increases. In Example 1, when the amount of amphiphilic polymer and EGCG is 3%, the storage stability of the product is optimal. Since the encapsulation efficiency and stability effects of the active ingredient EGCG and amphiphilic polymer used in Examples 11-13 and Comparative Example 2 are relatively poor, in order to save raw materials, the other effects and performances of Examples 11-13 and Comparative Example 2 will not be explored later.

[0087] Comparing Example 1 with Comparative Examples 3-7, it can be seen that: comparing Comparative Examples 3 and 6, the dialysis removal of polyols and the dissolution preparation method of the present invention have a greater impact on the stability of the nanocomposition; and compared with the use of liposomes (Comparative Example 4), nanoemulsification (Comparative Example 5) and a single aqueous solution dissolution method, the storage stability of the amphiphilic polymer encapsulation strategy of the present invention is significantly better.

[0088] Effect Example 2 In order to explore the transdermal absorption effect of the deep antioxidant EGCG nanocomposition provided by the present invention, a transdermal absorption (permeation enhancement) experiment was used to evaluate the transdermal absorption (permeation enhancement) ability of the nanocomposition or other products in the examples and comparative examples. The specific method is as follows: In vitro transdermal experiments used a vertical diffusion cell and nude mouse skin as a model (abdominal skin, subcutaneous fat layer and blood vessels removed). The receiving solution was a PBS solution. The skin sheet was fixed between the supply cell and the receiving cell, with the skin layer facing up, and balanced for 20 minutes. The nanocomposite samples in each embodiment and comparative example were formulated into a sample solution with a mass percentage of 10%, and the solution was added to the supply cell, and the receiving solution was taken after 1h, 4h, 8h and 24h. The receiving solution was ultrasonically demulsified with a mixed solution of methanol and deionized water, and ultrasonicated for 30 minutes according to the receiving solution: methanol: water = 1:4:5 (V / V). After filtering with a 0.45μm organic filter membrane, the EGCG content was determined by high performance liquid chromatography (HPLC, Shimadzu, Japan), and the cumulative permeation per unit area was calculated based on this. Three parallel experiments were performed for each group of experiments, and the arithmetic mean of the experimental results was taken. The calculation formula for the cumulative permeation per unit area on the skin sheet is as follows: Among them, Q n is the cumulative transmittance per unit area of ​​the sample at time t (μg / cm 2 ), A is the permeable area, C n is the concentration of the active substance measured at time t, C i is the concentration of the active ingredient measured at time point t, V is the total volume of the receptor solution, and V0 is the sampling volume at each time point. The results of the in vitro transdermal test are shown in Table 2 below.

[0089] Table 2 In vitro transdermal test results of Examples and Comparative Examples From Table 2 we can see that: Comparing Examples 1-5, it can be seen that the different degrees of polymerization of the hydrophobic block PGA and the hydrophilic block PMPC in the amphiphilic polymer used to prepare the nanocomposite will affect the transdermal permeability of the product nanocomposite. As the degree of polymerization of the hydrophobic block PGA or the hydrophilic block PMPC in the amphiphilic polymer increases, the transdermal permeability of the nanocomposite first increases and then decreases. In Example 1, when the degree of polymerization of the hydrophobic block PGA in the amphiphilic polymer is 60 and the degree of polymerization of the hydrophilic block PMPC is 120, the cumulative permeation per unit area of ​​EGCG in the final product at 1h, 4h, 8h and 24h is 6.42μg / cm 2 , 21.47μg / cm 2 、36.15μg / cm 2 and 98.20 μg / cm 2 , which are significantly higher than the nanocomposites of Examples 2-5, have the best transdermal penetration performance and are more conducive to achieving deep antioxidant effects.

[0090] Comparison of Examples 1, 6, and 7 with Comparative Example 1 shows that the addition of stabilizers and their types affect the transdermal permeability of the nanocomposition. In Example 1, when phytosphingosine is selected as the stabilizer and interacts with the other components, the transdermal permeability is optimal, which is conducive to achieving deep antioxidant effects.

[0091] Comparison of Examples 1, 8-10 shows that the different amounts of active ingredient EGCG and amphiphilic polymer affect the transdermal permeability of the nanocomposition. In Example 1, the amount of amphiphilic polymer is 3%, and the transdermal permeability is the best.

[0092] By comparing Example 1 with Comparative Examples 3-7, it can be seen that: compared with Comparative Examples 3 and 6, the dialysis removal of polyols and the dissolution preparation method of the present invention have a significant effect on the transdermal permeability of the nanocomposition; and compared with the use of liposomes (Comparative Example 4), nanoemulsification (Comparative Example 5) and a single aqueous solution dissolution method, the present invention adopts an amphiphilic polymer encapsulation strategy to greatly improve the transdermal permeability of the active substance EGCG in the nanocomposition, providing a basis for achieving deep antioxidant.

[0093] Effect Example 3 In order to explore the cell affinity of the deep antioxidant nanocomposition provided by the present invention, an in vitro cytotoxicity test was used to evaluate the cell affinity of the nanocompositions or other products in the examples and comparative examples. The specific method is as follows: Human immortalized keratinocytes (HaCaT cells) were used as human skin tissue model cells, and the CCK-8 method was used to evaluate the in vitro cytotoxicity of the samples.

[0094] HaCaT cells in the logarithmic growth phase (density of 3000 cells / well) were seeded into a 96-well plate, and DMEM medium (containing 10% FBS and 1% P / S) was added. The HaCaT cells were placed in a cell culture incubator at 37°C and 5% CO2 for 24 hours. The culture medium was removed and replaced with a culture medium solution containing the test sample (the test concentration was set to 0.5%, 1.0%, 3.0% and 5.0%), and the cells were further cultured in the cell culture incubator for 48 hours. CCK-8 reagent (10 μL / well) was added to the 96-well plate and incubated for another 4 hours. The absorbance of the solution in each well at 450 nm was detected by an enzyme reader, and the survival rate of the HaCaT cells was calculated using the following formula: Among them, A 样品 Indicates the absorbance of the experimental group; A 对照 represents the absorbance of the control group; A 空白 The results of the in vitro cytotoxicity test are shown in Table 3.

[0095] Table 3 In vitro cytotoxicity test results of Examples and Comparative Examples From Table 3 we can see that: Comparing Examples 1-5, it can be seen that the different degrees of polymerization of the hydrophobic block PGA and the hydrophilic block PMPC in the amphiphilic polymer used to prepare the nanocomposite have an impact on the cell affinity of the nanocomposite of the present invention. As the degree of polymerization of the hydrophobic block PGA increases or the degree of polymerization of the hydrophilic block PMPC increases, the cell affinity first increases and then decreases. In Example 1, when the degree of polymerization of PGA in the amphiphilic polymer is 60 and the degree of polymerization of PMPC is 120, after incubation with HaCaT cells for 48 hours, the cell viability of samples with mass concentrations of 0.5%, 1.0%, 3.0%, and 5.0% was 104.2%, 103.3%, 101.4%, and 97.6%, respectively, showing excellent cell affinity. At a treatment concentration of 5%, the cell survival rate was significantly higher than that of the samples in Examples 2-5, indicating that Example 1 has the best cell affinity and is more conducive to its efficacy at the cellular level.

[0096] Comparison of Examples 1, 6, and 7 with Comparative Example 1 shows that the addition of a stabilizer and its type affect the cell affinity of the nanocomposite. When phytosphingosine was selected as the stabilizer, the cell survival rate was the highest and the cell affinity was optimal, which is more conducive to its efficacy at the cellular level.

[0097] Comparing Example 1 and Examples 8-10, as the amount of amphiphilic polymer increased, the cell affinity of the nanocomposition first increased and then weakened; when the addition amount of the amphiphilic polymer was 3%, the cell survival rate and cell affinity of the nanocomposition were the highest after co-incubation with HaCaT cells, which was more conducive to its efficacy at the cellular level.

[0098] By comparing Example 1 with Comparative Examples 3-7, it can be seen that: comparing Comparative Examples 3 and 6, the dialysis removal of polyols and the dissolution preparation method of the present invention have a greater effect on the cell affinity of the nanocomposition (especially at higher concentrations); and compared with the use of liposomes (Comparative Example 4), nanoemulsification (Comparative Example 5) and a single aqueous solution dissolution method, the present invention adopts an amphiphilic polymer encapsulation strategy, which greatly reduces the cytotoxicity of the nanocomposition during the action process and greatly improves the cell survival rate, which is beneficial to exerting efficacy at the cellular level.

[0099] Effect Example 4 To preliminarily explore the antioxidant efficacy of the deep antioxidant nanocomposition provided by the present invention, the antioxidant properties of the nanocompositions or other products in the Examples and Comparative Examples were evaluated using a cell reactive oxygen species (ROS) inhibition test. The specific method is as follows: The samples of the embodiment and the comparative example were respectively prepared into 1% by mass solution using PBS buffer (pH 6.8).

[0100] Human skin keratinocytes (HaCaT) were digested and prepared into cell suspension, which was then seeded into 96-well plates at 1.0×10 5 -2.0×10 5 Cells were cultured in DMEM medium. After 24 hours of culture, the medium was replaced and a blank group (PBS buffer was added and no light was applied), a control group (PBS buffer was added and light was applied) and an experimental group (the example and comparative example samples were added, and the addition amount was 10% of the cell culture medium) were set up. The culture was continued for 24 hours, and then the control group and the experimental group were exposed to UVA light (the light dose was 6-10J / cm 2 After illumination, the cell culture medium was removed and the cells were washed three times with PBS. 100 μL of 10 μmol / L DCFH-DA was added to each well and incubated in a CO2 incubator for 20 min. After incubation, the cells were washed three times with PBS and serum-free DMEM was added again. ROS content was measured using a fluorescence microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The relative level of cellular ROS was calculated as the ratio of the OD value of the experimental group (or the OD value of the control group) to the OD value of the blank group. The results are shown in Table 4.

[0101] Table 4 Relative ROS levels in HaCaT cells after light damage after sample treatment sample Relative level of cellular ROS (%) Example 1 101.5 Example 2 126.5 Example 3 122.6 Example 4 123.2 Example 5 134.6 Example 6 120.5 Example 7 123.9 Example 8 138.5 Example 9 132.0 Example 10 127.5 Comparative Example 1 153.6 Comparative Example 3 154.6 Comparative Example 4 152.2 Comparative Example 5 159.5 Comparative Example 6 158.7 Comparative Example 7 150.1 Blank group 100.0 control group 181.5 From Table 4 we can see that: The samples prepared from the deep antioxidant EGCG nanocompositions provided in Examples 1-10 of the present invention had lower relative ROS levels in HaCaT cells damaged by light than those in the control group. The nanocompositions obtained by the preparation method of the present invention have good antioxidant properties and can effectively remove reactive oxygen species in cells.

[0102] Moreover, the relative levels of ROS in the HaCaT cells damaged by light after the samples prepared from the nanocompositions of Examples 1-10 were significantly lower than those in Comparative Example 1 and Comparative Examples 3-6, which indicates that compared with other EGCG nanocomposition preparations or other encapsulation strategies, the nanocompositions of the embodiments within the scope of the present invention have better antioxidant properties.

[0103] A horizontal comparison of the relative levels of cellular ROS in each example shows that the degree of polymerization of the hydrophobic block PGA and the hydrophilic block PMPC of the amphiphilic polymer used in the preparation step, the amount of amphiphilic polymer used, the addition of the stabilizer and its type all affect the antioxidant properties of the nanocomposition provided by the present invention. This is related to the combined influence of the EGCG encapsulation rate, stability, transdermal absorption performance, cell affinity and the structure of the nanocomposition itself. Among them, the deep antioxidant nanocomposition prepared by the strategy adopted in Example 1 has the lowest relative level of ROS in HaCaT cells after light damage. After co-incubation with the HaCaT cells after light damage for 24 hours, the relative level of ROS is 102.5%, which is close to the level of normal cells without light damage. The nanocomposition prepared by the preparation strategy of Example 1 has the best antioxidant properties.

[0104] In addition, it can be seen that compared with Comparative Example 7, the nanocomposition in Example 1 has a significantly lower relative level of ROS in HaCaT cells after light damage, which indicates that compared with the EGCG monomer without encapsulation treatment, the amphiphilic polymer and specific stabilizer used in the present invention are combined to encapsulate the active substance EGCG, and the resulting nanocomposition can exert a better antioxidant effect.

[0105] Effect Example 5 To further explore the deep antioxidant efficacy of the deep antioxidant EGCG nanocomposition provided by the present invention, the antioxidant signaling pathways of the nanocomposition of Example 1, which is the best among the above-mentioned effects, and other products in Comparative Examples 1, 3-7 were evaluated experimentally. The specific methods are as follows: When the stratum corneum is damaged or destroyed, the skin barrier is compromised, leading to a hypermetabolic response, weakened cellular antioxidant mechanisms, and damaged antioxidant systems. Oxidative stress, a key factor in skin barrier repair, generates large amounts of ROS free radicals, which damage proteins, lipids, and DNA in the skin tissue, thereby reducing cellular physiological activity, disrupting normal signaling pathways, and exacerbating inflammatory responses. H2O2 is an oxidant that diffuses freely between and within cells. Excessive concentrations can cause irreversible oxidative damage to cells, and it is often used as an oxidative damage model.

[0106] HaCaT cells in the logarithmic growth phase were taken and 2.5×10 5 2 mL of cells were plated in 6-well plates at a cell density of 5 × 10 cells / mL. 5 The cells were plated and cultured in a cell culture incubator for 12 h. The original culture medium was discarded and a culture medium containing the sample was added (the mass percentage of the product of each embodiment and comparative example in the sample was 0.5%). The cells were pretreated for 12 h. After 12 h, 100 μM H2O2 was added to each well and the cells were incubated for 12 h. The culture medium was aspirated and the cells were washed twice with fresh PBS solution.

[0107] Total RNA was extracted using the Simply P Total RNA Extraction Kit (Shanghai Biotech Co., Ltd.) according to the manufacturer's instructions. The obtained total RNA was then tested for purity.

[0108] The extracted total RNA was reverse transcribed using the RT Master Mix for qPCR (gDNA digester plus, Shanghai Beyotime Biotechnology Co., Ltd.) kit to obtain cDNA.

[0109] Calculate the amount of RNA based on the extracted RNA concentration, and add the following reagents in sequence to a 10 μL reaction system in an RNase-free PCR tube: 2 μL of 5× gDNA digester Buffer, 1 μL of gDNA digester, 5 μL of Total RNA, and 2 μL of RNase-Free H2O. Then, add 2× Super RT Mix to each reaction tube, pipette to mix, and place in a PCR instrument for reaction at 25°C for 5 min, 42°C for 40 min, and 85°C for 2 min.

[0110] GAPDH was selected as the internal reference gene and synthesized according to the primer gene sequence in Table 5.

[0111] NovoStart SYBR quantitative reagent and primers were prepared according to a 20 μL reaction system: 2×NovoStartSYBR qPCR SuperMix Plus (10 μL), upstream primer (0.2 μL), downstream primer (0.2 μL), cDNA (2 μL), and RNase-Free Water (7.6 μL).

[0112] According to the required amount for each group, the quantitative reagents and primers were mixed in advance. After the template was mixed with RNase-free water, they were added to the 96-well plate of fluorescent quantitative PCR in sequence. The fluorescent quantitative membrane was tightly attached to the 96-well plate to reduce the evaporation loss of each reagent during the reaction. Then, the plate was centrifuged at 4°C and 3000 rpm for 3 minutes before running on the machine.

[0113] Table 5 Primer gene sequences The relative expression levels of the antioxidant NQO1 gene, CAT gene, Nrf2 gene, SOD1 gene, and SOD2 gene in HaCaT cells pretreated with the nanocomposition or other products in Example 1 and Comparative Examples 1, 3-7 for oxidative damage repair are as follows: Figure 3 、 4 , 5, 6, and 7.

[0114] Depend on Figure 3-7 It can be seen that the nanocomposition prepared by the preparation strategy of Example 1 can upregulate the NQO1, CAT, Nrf2, SOD1, and SOD2 genes in the cells after co-incubation with HaCaT cells damaged by H2O2, and the upregulation is 5.12 times, 9.67 times, 5.83 times, 11.4 times, and 11.0 times, respectively; Overall, the nanocomposition prepared by the preparation strategy in Example 1 of the present invention has excellent antioxidant properties, which can significantly mobilize the antioxidant defense mechanism in the cells, initiate oxidative damage repair, and enhance the expression of antioxidant-related enzymes; through good cell affinity and transdermal absorption delivery, it can achieve the promotion of deep-layer antioxidant activity in cells.

[0115] At the same time, it can be seen that the upregulation of NQO1, CAT, Nrf2, SOD1, and SOD2 genes is better than that of Comparative Examples 1 and 3-7. Compared with the nanocompositions prepared by adjusting the preparation steps in Comparative Examples 3 and 6, the liposomes or nanoemulsions in Comparative Examples 4 and 5, and the single EGCG solution without entrapment treatment in Comparative Example 7, the present invention uses a specific amphiphilic polymer and a specific stabilizer to encapsulate the active substance EGCG. The prepared deep antioxidant EGCG nanocomposition can better achieve deep antioxidant performance under high entrapment rate, stability, cell affinity and transdermal absorption, and achieve comprehensive and deeper antioxidant efficacy by effectively regulating the expression of antioxidant-related genes.

[0116] In summary, the present invention optimizes amphiphilic polymers, selects specific hydrophobic segments PGA and hydrophilic segments PMPC as carriers of active ingredients, improves the transdermal absorption effect and cell affinity of the nanocomposition; self-assembles the amphiphilic polymers with specific amounts of phytosphingosine stabilizers and antioxidant active ingredients ECGG under specific preparation steps to obtain a nanocomposition and improve the entrapment rate and stability of EGCG; by optimizing the preparation steps, the present invention prepares a deep antioxidant nanocomposition that can significantly improve the transdermal absorption of EGCG, has high cell affinity and deep antioxidant efficacy. The preparation strategy of the present invention solves the problems of poor stability, poor transdermal absorption, and inability of EGCG to enter the skin cells to exert deep antioxidant efficacy in the prior art, achieves deep antioxidant efficacy at the cellular level, and has high application value.

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A deep antioxidant EGCG nanocomposition, characterized in that: The raw materials for preparing the deep antioxidant EGCG nanocomposition include a carrier, a stabilizer, an active substance, water, and a polyol; The active substance is EGCG; the mass percentage of EGCG in the raw materials for preparing the deep antioxidant EGCG nanocomposition is 0.5-5%; The stabilizer is at least one of phytosphingosine and its derivatives; The carrier is an amphiphilic polymer, and the general formula of the amphiphilic polymer is X m -Y n , the X m is polyglycolide, said Y n It is poly 2-methacryloyloxyethyl phosphorylcholine.

2. The deep antioxidant EGCG nanocomposition according to claim 1, characterized in that The preparation method of the amphiphilic polymer comprises the following steps: S1. Mix the initiator, glycolide and catalyst, and react at 80-150° C. for 6-24 hours to obtain polyglycolide; S2, adding the polyglycolide and the bromination reagent to a solvent and mixing them evenly, and reacting them at 0-25° C. for 12-36 hours to obtain brominated polyglycolide; S3, adding the brominated polyglycolide, 2-methacryloyloxyethyl phosphorylcholine, catalyst and catalyst ligand to the solvent, mixing, and reacting at 60-100° C. for 24-72 hours to obtain the amphiphilic polymer; the reactions in S1, S2 and S3 are all carried out under an inert atmosphere.

3. The deep antioxidant EGCG nanocomposition according to claim 2, characterized in that In step S1, the catalyst is at least one of stannous octoate, stannous isooctanoate, stannous tartrate, and stannous stearate, and the initiator is a C1-C6 monohydric alcohol; and / or, in step S1, the molar ratio of the initiator, glycolide, and catalyst is initiator:glycolide:catalyst=1:(20-40):(2-4); And / or, in step S2, the bromination reagent is at least one of 2-bromoisobutyryl bromide, tert-butyl 2-bromoisobutyrate, and ethyl 2-bromoisovalerate; And / or, in step S2, the molar ratio of polyglycolide to the bromination reagent is 1:(2-8); And / or, in step S3, the catalyst is at least one of CuBr, CuCl, and CuI, and the catalyst ligand is an amine or ammonium catalyst ligand; And / or, in step S3, the molar ratio of brominated polyglycolide, 2-methacryloyloxyethyl phosphorylcholine, catalyst, and catalyst ligand is brominated polyglycolide:2-methacryloyloxyethyl phosphorylcholine:catalyst:catalyst ligand=1:(80-160):(0.8-1.2):(8-16).

4. The deep antioxidant EGCG nanocomposition according to claim 1, characterized in that The degree of polymerization of the polyglycolide is m=40-80; And / or, the degree of polymerization of the poly (2-methacryloyloxyethyl) phosphorylcholine is n=80-160.

5. The deep antioxidant EGCG nanocomposition according to claim 1, characterized in that The stabilizer is at least one of phytosphingosine, tetraacetyl phytosphingosine, and phytosphingosine salicylate; And / or, the polyol is C2-C 10 Polyols.

6. The deep antioxidant EGCG nanocomposition according to claim 1, characterized in that The raw materials for preparing the deep antioxidant EGCG nanocomposition include 0.5-5% by mass of the amphiphilic polymer, 0.5-5% by mass of EGCG, 0.05-1% by mass of the stabilizer, 5-30% by mass of the polyol, and the balance being water.

7. The deep antioxidant EGCG nanocomposition according to claim 4, characterized in that The polymerization degree of the polyglycolide is m=50-70, and the polymerization degree of the poly-2-methacryloyloxyethyl phosphorylcholine is n=100-140; And / or, in the raw materials for preparing the deep antioxidant EGCG nanocomposition, the mass percentage of the amphiphilic polymer is 2-4%, the mass percentage of EGCG is 2-4%, the mass percentage of the stabilizer is 0.2-0.5%, the mass percentage of the polyol is 20-30%, and the balance is water.

8. The method for preparing the deep antioxidant EGCG nanocomposition according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) heating and mixing the amphiphilic polymer, stabilizer, and EGCG in a polyol to obtain an alcohol phase solution; (2) adding the alcohol phase solution dropwise to water while subjecting it to high shear treatment to obtain a mixed solution; (3) removing the polyol from the mixed solution and then homogenizing it to obtain the deep antioxidant EGCG nanocomposite.

9. The method for preparing the deep antioxidant EGCG nanocomposition according to claim 8, wherein: In the step (1), the temperature of heating and mixing is 35-80°C; And / or, in the step (2), the rate of adding the alcohol phase solution is 1-10 mL / min, the rotation speed of the high shear treatment is 6000-12000 rpm, and the high shear treatment is performed until the addition of the alcohol phase solution is completed; And / or, in step (3), dialysis is used to remove the polyol, and the pore size of the dialysis bag is 300-1000Da; And / or, the pressure of the homogenization treatment in step (3) is 50-120 MPa, and the number of homogenization cycles is 2-8 times.

10. Use of the deep antioxidant EGCG nanocomposition according to any one of claims 1 to 7 in antioxidant cosmetics.

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