A deep antioxidant egcg nano composition and its preparation method and use

Abstract

The present application relates to a kind of deep antioxidant EGCG nano composition and its preparation method and purposes, belong to nanocarrier technical field.Deep antioxidant EGCG nano composition of the present application preparation raw material includes carrier, stabilizer, active substance, water, polyhydric alcohol;Active substance is EGCG;The mass percentage of EGCG in preparation raw material is 0.5-5%;Stabilizer is selected from plant sphingosine and its derivatives;Carrier is amphiphilic polymer, general formula is X m -Y n , wherein X m It is polyglycolide, Y n It is poly 2-methacryloyloxy ethyl phosphorylcholine.The nano composition is prepared by removing polyhydric alcohol in raw material after self-assembly by specific preparation method.The nano composition provided by the present application has high encapsulation efficiency, good stability, and good transdermal absorption, and is used in cosmetics, which can provide excellent deep antioxidant effect on skin.

Description

Technical Field

[0001] This invention relates to the field of nanocarrier technology, and in particular to a deep antioxidant EGCG nanocomposition, its preparation method, and its uses. Background Technology

[0002] Epigallocatechin gallate (EGCG) is a component extracted from Chinese green tea. It is the main active ingredient in green tea and the most abundant catechin component, accounting for 9-13% of the gross weight of green tea. EGCG has a unique stereochemical structure and very strong antioxidant activity. Its molecular structure contains 8 phenolic hydroxyl groups and 2 benzodihydropyran rings, giving it a very strong ability to scavenge oxygen free radicals. It can protect cells and DNA from damage, effectively neutralize ROS / RNS through electron transfer and proton transfer mechanisms, and chelate metal ions to block oxidation chain reactions.

[0003] EGCG can be used as an antioxidant active ingredient to combat skin oxidation and aging. However, there are three major bottlenecks in the practical application of EGCG in the cosmetic field: ① In terms of stability, because the molecular structure of EGCG is rich in phenolic hydroxyl groups, these hydroxyl groups are easily oxidized to form red quinone derivatives under light (especially UVB), high temperature (>40℃), and pH>6 environments, and the antioxidant effect is correspondingly weakened, leading to product browning and an activity decay rate as high as 80%; ② In terms of transdermal absorption, the high water solubility of EGCG conflicts with the lipid barrier of the stratum corneum (composed of ceramides, cholesterol, etc., which are lipid-soluble), often remaining only on the outermost part of the stratum corneum and unable to effectively penetrate the stratum corneum to better exert its effects. Conventional formulas have very low transdermal rates, requiring the use of nanoliposomes or microemulsion carriers to improve delivery efficiency; ③ In terms of bioavailability, skin surface metabolic enzymes can rapidly degrade EGCG.

[0004] To address the problems existing in the addition of EGCG to cosmetics and to broaden its applications, there is an urgent need to develop a strategy that can improve the stability and transdermal absorption of EGCG and realize its deep antioxidant function on the skin. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a deep-penetrating antioxidant EGCG nanocomposition, its preparation method, and its uses. The nanocomposition of this invention exhibits high encapsulation efficiency for EGCG, good storage stability, high transdermal absorption, and cell affinity, while also providing excellent deep-penetrating antioxidant effects.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] 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;

[0008] The active substance is EGCG; in the raw materials for preparing the deep antioxidant EGCG nanocomposition, the mass percentage of EGCG is 0.5-5%;

[0009] The stabilizer is at least one of phytosphingosine and its derivatives;

[0010] The carrier is an amphiphilic polymer, and the general formula of the amphiphilic polymer is X. m -Y n The X m It is polyglycolic acid, and the Y is... n It is poly(2-methacryloyloxyethylphosphorylcholine).

[0011] This invention utilizes an amphiphilic polymer with specific hydrophobic and hydrophilic segments and the antioxidant active ingredient ECGG to achieve self-assembly during the preparation process with a specific type of stabilizer, resulting in a deep antioxidant EGCG nanocomposition that can significantly improve the transdermal absorption of EGCG, enhance encapsulation efficiency and stability, and simultaneously possess high cell affinity and deep antioxidant efficacy.

[0012] The amphiphilic polymer used has a hydrophobic segment composed of polyglycolic acid (PGA). The strong intermolecular forces of PGA can enhance the physical stability of the formed nanocomposite, and its suitable hydrophobicity can promote the compatibility of the nanocomposite 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 with a degradation rate that matches the cell metabolic cycle, which can reduce cell irritation, has high cell affinity, and good biocompatibility.

[0013] The hydrophilic segment of the amphiphilic polymer is composed of poly(2-methacryloyloxyethylphosphorylcholine) (PMPC), which is an amphoteric polymer with both positive and negative charged groups present in the molecule. This can reduce the aggregation of the nanocomposite through an electrostatic shielding effect, thus improving stability. Simultaneously, PMPC enhances the compatibility with the skin's lipid layer (mainly composed of ceramides and cholesterol), strengthening the intercellular lipid pathway of the stratum corneum and promoting the penetration of the nanocomposite. Combining the hydrophilic and hydrophobic segments, this invention utilizes the PMPC hydrophilic segment of the amphiphilic polymer to encapsulate and stabilize the catechol groups of EGCG, thereby improving its antioxidant activity retention and reducing its contact with external moisture, oxygen, and metal ions, thus comprehensively improving the antioxidant efficacy of EGCG when used in cosmetics.

[0014] By selecting specific stabilizers, phytosphingosine and its derivatives, the lipid portion can bind to the hydrophobic segment of the amphiphilic polymer through hydrophobic interactions, forming more stable micelles and enhancing stability. Phytosphingosine substances can also reduce the metabolic loss of nanocomposites 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 nanocomposites. Through structural interactions and enhanced membrane affinity with amphiphilic polymers, they can significantly improve the transdermal efficiency and stability of nanocomposites and enhance deep antioxidant effects.

[0015] This invention encapsulates a specific amount of EGCG using an amphiphilic polymer, PGA (hydrophobic segment) and PMPC (hydrophilic segment), and adds a specific stabilizer to further improve performance. This effectively enhances the stability, transdermal permeability, cell affinity, and antioxidant properties of EGCG. Compared to free EGCG or commonly used liposome formulations and nanoemulsions, the nanocomposition of this invention exhibits higher stability, better transdermal permeability, and superior antioxidant efficacy. It can act deep into skin cells through deep delivery, effectively regulating the expression of antioxidant genes and achieving comprehensive and deeper antioxidant effects.

[0016] Preferably, the method for preparing the amphiphilic polymer includes the following steps:

[0017] S1, mix the initiator, glycolide and catalyst, and react at 80-150℃ for 6-24h to obtain polyglycolic acid;

[0018] S2. Add the polyglycolic acid and the brominated reagent to the solvent and mix well. React at 0-25℃ for 12-36h to obtain brominated polyglycolic acid.

[0019] S3. The brominated polyglycolic acid, 2-methacryloyloxyethyl phosphorylcholine, catalyst and catalyst ligand are added to the solvent and mixed. The mixture is reacted at 60-100℃ for 24-72 h to obtain the amphiphilic polymer. The reactions in S1, S2 and S3 are all carried out under an inert atmosphere.

[0020] 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.

[0021] Preferably, in step S1, the molar ratio of initiator, glycolide, and catalyst is initiator: glycolide: catalyst = 1:(20-40):(2-4);

[0022] Preferably, in step S2, the brominating agent is at least one selected from 2-bromoisobutyryl bromide, tert-butyl 2-bromoisobutyrate, and ethyl 2-bromoisovalerate.

[0023] Preferably, in step S2, the molar ratio of polyglycolic acid to brominated reagent is 1:(2-8);

[0024] 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.

[0025] Preferably, in step S3, the molar ratio of brominated polyglycolic acid, 2-methacryloyloxyethyl phosphorylcholine, catalyst, and catalyst ligand is brominated polyglycolic acid: 2-methacryloyloxyethyl phosphorylcholine: catalyst: catalyst ligand = 1:(80-160):(0.8-1.2):(8-16).

[0026] More preferably, the initiator is at least one selected from methanol, ethanol, n-propanol, 2-propanol, n-butanol, 2-butanol, n-pentanol, 2-pentanol, 3-pentanol, n-hexanol, 2-hexanol, and 3-hexanol.

[0027] More preferably, the catalyst ligand is at least one selected from N,N-diisopropylethylamine, ethanolamine, N,N,N',N',N”-pentamethyldiethylenetriamine, and tetrabutylammonium bromide.

[0028] More preferably, the solvent is a solvent commonly used in the art, and may be selected from at least one of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, dichloromethane, tetrahydrofuran, acetone, chloroform, acetonitrile, and ethyl acetate.

[0029] More preferably, the inert gas is an inert gas conventionally used in the art, and can be selected from one or more of nitrogen, argon, and helium, with argon being preferred.

[0030] More preferably, after the reaction of the amphiphilic polymer is completed, a purification process is also included. The purification process specifically involves: slowly adding the amphiphilic polymer to a precipitant, performing precipitation purification, filtering, washing the solid multiple times with the precipitant, and drying at 25-80℃ for 12-72 hours to obtain a purified amphiphilic polymer; the precipitant includes at least one of methanol, ethanol, n-hexane, and petroleum ether; the precipitation purification temperature is -10 to 10℃.

[0031] In the preparation steps of the amphiphilic polymer, the hydrophobic segment polyglycolic acid (PGA) of the amphiphilic polymer is obtained from the polymer monomer glycolide through ring-opening polymerization (ROP); the hydrophilic block poly(2-methacryloyloxyethylphosphorylcholine) (PMPC) of the amphiphilic polymer is obtained from the polymer monomer 2-methacryloyloxyethylphosphorylcholine through atom transfer radical polymerization (ATRP); finally, the hydrophobic and hydrophilic segments are connected by reaction under the action of initiator molecules to obtain the amphiphilic polymer.

[0032] Preferably, the degree of polymerization of the polyglycolic acid is m = 40-80;

[0033] Preferably, the degree of polymerization of the poly(2-methacryloyloxyethylphosphorylcholine) is n = 80-160.

[0034] Preferably, the stabilizer is at least one of phytosphingosine, tetraacetylphytosphingosine, and salicylic acid phytosphingosine;

[0035] Preferably, the polyol is C2-C 10 Polyols.

[0036] More preferably, the polyol is at least one selected from 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, 1,2-butanediol, 1,4-butanediol, dipropylene glycol, 1,2-hexanediol, 1,6-hexanediol, ethoxydiethylene glycol, 1,8-octanediol, and 1,10-decanediol.

[0037] More preferably, the stabilizer is phytosphingosine.

[0038] Different stabilizer choices affect the storage stability, transdermal permeability, and cell affinity of the nanocompositions prepared in this invention. Using preferred stabilizers can significantly improve the overall performance of the nanocompositions.

[0039] Preferably, in the raw materials for preparation, 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.

[0040] Preferably, the mass percentage of the amphiphilic polymer in the raw materials is one or any two of the following: 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, and 5.0%.

[0041] Preferably, the mass percentage of EGCG in the raw materials is one or any two of the following: 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, and 5.0%.

[0042] More preferably, the degree of polymerization of the polyglycolic acid is m = 50-70, and the degree of polymerization of the poly(2-methacryloyloxyethylphosphorylcholine) is n = 100-140.

[0043] Preferably, in the raw materials for preparation, 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.

[0044] The ratio of amphiphilic polymers and EGCG components during the preparation of nanocomposites, as well as the degree of polymerization of the hydrophobic and hydrophilic segments in the amphiphilic polymers, will ultimately affect the storage performance, transdermal absorption performance, cell affinity, and antioxidant efficacy of the nanocomposites during use. Under the defined preferred degree of polymerization of the hydrophobic and hydrophilic segments, the nanocomposites have superior cell affinity, which is more conducive to their efficacy at the cellular level.

[0045] Secondly, the present invention provides a method for preparing the above-mentioned deep antioxidant EGCG nanocomposition, comprising the following steps:

[0046] (1) The amphiphilic polymer, stabilizer and EGCG are heated and mixed in a polyol to obtain an alcohol phase solution;

[0047] (2) The alcohol phase solution is added dropwise to water and subjected to high shear treatment to obtain a mixed solution;

[0048] (3) Remove the polyol from the mixed solution and then homogenize it to obtain the deep antioxidant EGCG nanocomposition.

[0049] In the preparation process, the alcohol phase solution is initially prepared into self-assembled nanomaterials after high shear treatment. At this time, it is necessary to remove the polyol. The remaining nanocomposite forms a more uniform dispersion system through hydrogen bonding and van der Waals forces, which enhances the interfacial bonding force 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.

[0050] Preferably, in step (1), the temperature for heating and mixing is 35-80°C;

[0051] Preferably, in 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 continues until the alcohol phase solution is added.

[0052] Preferably, in step (3), dialysis is used to remove polyols, and the pore size of the dialysis bag is 300-1000 Da;

[0053] Preferably, the homogenization pressure in step (3) is 50-120 MPa, and the number of homogenization cycles is 2-8.

[0054] This invention employs dialysis bags with specific pore sizes for dialysis operations, ensuring that the molecular weight cutoff of the dialysis bag is greater than that of the polyol, and the pore size is much smaller than the particle size of the nanocomposite. This allows for the successful removal of polyols from the solution while retaining the prepared deep-penetrating antioxidant EGCG nanocomposite.

[0055] As a preferred embodiment of the present invention, in step (1), the stirring speed for heating and mixing is 200-500 rpm; the stirring time is 1-6 h.

[0056] As a preferred embodiment of the present invention, in step (2), after the addition is completed, the mixture is stirred at 200-500 rpm for 1-6 hours.

[0057] As a preferred embodiment of the present invention, in step (3), the dialysis treatment time is 12-72 hours.

[0058] As a preferred embodiment of the present invention, the stirring method is mechanical stirring or magnetic stirring.

[0059] The deep antioxidant EGCG nanocomposition prepared by the above method can achieve an encapsulation rate of over 93% for EGCG and exhibits good stability. It also has good transdermal absorption, high cumulative permeation per unit area, and better cell affinity, with an average cell survival rate of over 95% after long-term treatment at a 1% concentration. This deep antioxidant nanocomposition comprehensively enhances antioxidant performance, upregulates intracellular NQO1, CAT, Nrf2, SOD1, and SOD2 genes, and promotes the activation of antioxidant defense mechanisms at the cellular gene level, thereby achieving a deep antioxidant effect on skin cells.

[0060] Thirdly, the present invention provides the use of the above-mentioned deep antioxidant EGCG nanocomposition in antioxidant cosmetics.

[0061] Preferably, the use is to add the deep antioxidant EGCG nanocomposition to antioxidant cosmetics; the amount of the deep antioxidant EGCG nanocomposition added is 0.1-5% by mass percentage.

[0062] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0063] This invention optimizes amphiphilic polymers, selects specific hydrophobic and hydrophilic segments, and uses phytosphingosine and its derivatives as stabilizers to achieve self-assembly with the antioxidant active ingredient ECGG under specific preparation steps. This provides a deep-penetrating antioxidant EGCG nanocomposition that significantly improves transdermal absorption, enhances encapsulation efficiency and stability, and simultaneously possesses high cell affinity and deep-penetrating antioxidant effects. It solves the problems of poor stability, poor transdermal absorption, and inability to penetrate skin cells to exert deep-penetrating antioxidant effects in existing technologies, and has high application value. Attached Figure Description

[0064] Figure 1 The synthetic routes for the amphiphilic polymers in Examples 1-5 are shown below;

[0065] Figure 2 The GPC curves of the amphiphilic polymers in Examples 1-5 are shown below.

[0066] Figure 3 Figure showing the expression of the antioxidant NQO1 gene in HaCaT cells undergoing oxidative damage repair.

[0067] Figure 4 Figure showing the expression of the antioxidant CAT gene in HaCaT cells undergoing oxidative damage repair;

[0068] Figure 5 Figure showing the expression of the antioxidant Nrf2 gene in HaCaT cells undergoing oxidative damage repair;

[0069] Figure 6 Figure showing the expression of the antioxidant SOD1 gene in HaCaT cells undergoing oxidative damage repair.

[0070] Figure 7 Figure showing the expression of the antioxidant SOD2 gene in HaCaT cells undergoing oxidative damage repair. Detailed Implementation

[0071] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available reagents and materials.

[0072] In the embodiments and comparative examples of this invention, EGCG, phytosphingosine, tetraacetyl phytosphingosine, and salicylic acid phytosphingosine were purchased from Guangzhou Xiyuan Biotechnology Co., Ltd.

[0073] The synthetic route diagram of the amphiphilic polymer in the following examples is as follows: Figure 1 ;

[0074] The molecular weight of the amphiphilic polymers in the following examples was determined using a gel permeation chromatography (GPC, manufacturer: Waters Corporation, USA; model: Waters 1525 / 2414). Chromatographically pure THF solution was used as the mobile phase, the flow rate was 1.0 mL / min, the column was calibrated with a monodistributed polystyrene standard, and the test temperature was 30 °C.

[0075] Example 1

[0076] One embodiment of the deep antioxidant EGCG nanocomposition of the present invention, wherein the raw materials for preparing the deep antioxidant EGCG nanocomposition in this embodiment include the following components by mass percentage: 3% amphiphilic polymer carrier PGA 60 PMPC 120 0.3% stabilizer phytosphingosine, 3% active ingredient EGCG, 20% 1,3-butanediol, and the balance is water.

[0077] The amphiphilic polymer used in this embodiment is PGA. 60 PMPC 120 The preparation method is as follows (taking the preparation of 1.0 mmol as an example):

[0078] 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 thoroughly and reacted under argon protection at 130 °C for 12 h. The reaction mixture was then added dropwise 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 polyglycolic acid PGA. 60 .

[0079] S2, PGA 60 (3.48 g, 1.0 mmol) was dissolved in tetrahydrofuran (100 mL) under argon protection, and 2-bromoisobutyryl bromide (0.92 g, 4.0 mmol) was added. The reaction mixture was reacted at 20 °C for 24 h. The reaction mixture was then added to excess petroleum ether (5 °C), and after precipitation purification, the solid product was collected and dried at 50 °C for 24 h to obtain brominated PGA. 60 .

[0080] S3, brominated PGA 603.61 g (1.0 mmol), 2-methacryloyloxyethylphosphorylcholine (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, CuBr (168 mg, 1.2 mmol) was added as a catalyst, and the reaction was carried out at 65 °C for 48 h. The reaction mixture was then added to excess petroleum ether (5 °C), and after precipitation 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 shown) Figure 2 Calculate Mn = 37812).

[0081] The preparation method of the deep antioxidant EGCG nanocomposition described in this embodiment is as follows:

[0082] (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 hours to obtain an alcohol phase solution.

[0083] (2) The alcohol phase solution was added dropwise to deionized water at a rate of 5 mL / min, and high-speed shearing was started simultaneously at a speed of 8000 rpm. After the addition was completed, the mixture was stirred at 400 rpm for 3 hours to obtain a mixed solution.

[0084] (3) Transfer the mixed solution to a dialysis bag (the molecular weight cutoff of the dialysis bag is 500 Da), and dialyze it in 10 times the volume of water of the mixed solution for 48 h, replacing the 1,3-butanediol in the mixed solution with deionized water, and changing the deionized water every 6 h.

[0085] After dialysis, the solution in the dialysis bag was taken, homogenized by microfluidic high pressure, and the pressure was adjusted to 70 MPa. This process was repeated 5 times to obtain the deep antioxidant EGCG nanocomposite.

[0086] Example 2

[0087] This invention provides an embodiment of the deep antioxidant EGCG nanocomposition. The only difference between this embodiment and Example 1 is that the amphiphilic polymer used as the carrier is PGA. 40 PMPC 120The 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, and reacted at 130 °C for 12 h under argon protection; then the reaction mixture is added dropwise to excess petroleum ether (5 °C), and after precipitation purification, the solid product is collected and dried at 50 °C for 24 h to obtain polyglycolic acid PGA. 40 .

[0088] S2, PGA 40 (2.32 g, 1.0 mmol) was dissolved in tetrahydrofuran (100 mL) under argon protection, and 2-bromoisobutyryl bromide (0.92 g, 4.0 mmol) was added. The reaction mixture was reacted at 20 °C for 24 h. The reaction mixture was then added to excess petroleum ether (5 °C), and after precipitation purification, the solid product was collected and dried at 50 °C for 24 h to obtain brominated PGA. 40 .

[0089] S3, brominated PGA 40 2.45 g (1.0 mmol), 2-methacryloyloxyethylphosphorylcholine (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, CuBr (168 mg, 1.2 mmol) was added as a catalyst, and the reaction was carried out at 65 °C for 48 h. The reaction mixture was then added to excess petroleum ether (5 °C), and after precipitation 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 shown) Figure 2 Calculate Mn = 36008).

[0090] The preparation method of the deep antioxidant EGCG nanocomposition described in this embodiment is the same as that in Example 1.

[0091] Example 3

[0092] This invention provides an embodiment of the deep antioxidant EGCG nanocomposition. The only difference between this embodiment and Example 1 is that the amphiphilic polymer used as the carrier is PGA. 80 PMPC 120The 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, and reacted at 130 °C for 12 h under argon protection; then the reaction mixture is added dropwise to excess petroleum ether (5 °C), and after precipitation purification, the solid product is collected and dried at 50 °C for 24 h to obtain polyglycolic acid PGA. 80 .

[0093] S2, PGA 80 (4.64 g, 1.0 mmol) was dissolved in tetrahydrofuran (100 mL) under argon protection, and 2-bromoisobutyryl bromide (0.92 g, 4.0 mmol) was added. The reaction mixture was reacted at 20 °C for 24 h. The reaction mixture was then added to excess petroleum ether (5 °C), and after precipitation purification, the solid product was collected and dried at 50 °C for 24 h to obtain brominated PGA. 80 .

[0094] S3, brominated PGA 80 4.77 g (1.0 mmol), 2-methacryloyloxyethylphosphorylcholine (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, CuBr (168 mg, 1.2 mmol) was added as a catalyst, and the reaction was carried out at 65 °C for 48 h. The reaction mixture was then added to excess petroleum ether (5 °C), and after precipitation 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 shown) Figure 2 Calculate Mn = 38457).

[0095] The preparation method of the deep antioxidant EGCG nanocomposition described in this embodiment is the same as that in Example 1.

[0096] Example 4

[0097] This invention provides an embodiment of the deep antioxidant EGCG nanocomposition. The only difference between this embodiment and Example 1 is that the amphiphilic polymer used as the carrier is PGA. 60 PMPC 80The 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, and reacted at 130 °C for 12 h under argon protection; then the reaction mixture is added dropwise to excess petroleum ether (5 °C), and after precipitation purification, the solid product is collected and dried at 50 °C for 24 h to obtain polyglycolic acid PGA. 60 .

[0098] S2, PGA 60 (3.48 g, 1.0 mmol) was dissolved in tetrahydrofuran (100 mL) under argon protection, and 2-bromoisobutyryl bromide (0.92 g, 4.0 mmol) was added. The reaction mixture was reacted at 20 °C for 24 h. The reaction mixture was then added to excess petroleum ether (5 °C), and after precipitation purification, the solid product was collected and dried at 50 °C for 24 h to obtain brominated PGA. 60 .

[0099] S3, brominated PGA 60 3.61 g (1.0 mmol), 2-methacryloyloxyethylphosphorylcholine (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, CuBr (112 mg, 0.8 mmol) was added as a catalyst, and the reaction was carried out at 65 °C for 48 h. The reaction mixture was then added to excess petroleum ether (5 °C), and after precipitation 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 shown) Figure 2 Calculate Mn = 25984).

[0100] The preparation method of the deep antioxidant EGCG nanocomposition described in this embodiment is the same as that in Example 1.

[0101] Example 5

[0102] This invention provides an embodiment of the deep antioxidant EGCG nanocomposition. The only difference between this embodiment and Example 1 is that the amphiphilic polymer used as the carrier 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, and reacted at 130 °C for 12 h under argon protection; then the reaction mixture is added dropwise to excess petroleum ether (5 °C), and after precipitation purification, the solid product is collected and dried at 50 °C for 24 h to obtain polyglycolic acid PGA. 60 .

[0103] S2, PGA 60 (3.48 g, 1.0 mmol) was dissolved in tetrahydrofuran (100 mL) under argon protection, and 2-bromoisobutyryl bromide (0.92 g, 4.0 mmol) was added. The reaction mixture was reacted at 20 °C for 24 h. The reaction mixture was then added to excess petroleum ether (5 °C), and after precipitation purification, the solid product was collected and dried at 50 °C for 24 h to obtain brominated PGA. 60 .

[0104] S3, brominated PGA 60 3.61 g (1.0 mmol), 2-methacryloyloxyethylphosphorylcholine (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). Under argon protection, CuBr (224 mg, 1.6 mmol) was added as a catalyst, and the reaction was carried out at 65 °C for 48 h. The reaction mixture was then added to excess petroleum ether (5 °C), and after precipitation 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 shown) Figure 2 Calculate Mn = 49157).

[0105] The preparation method of the deep antioxidant EGCG nanocomposition described in this embodiment is the same as that in Example 1.

[0106] Example 6

[0107] An embodiment of the deep antioxidant EGCG nanocomposition of the present invention differs from Example 1 only in that the stabilizer phytosphingosine is replaced with tetraacetyl phytosphingosine in the same mass content.

[0108] Example 7

[0109] An embodiment of the deep antioxidant EGCG nanocomposition of the present invention differs from Example 1 only in that the stabilizer phytosphingosine is replaced with salicylic acid phytosphingosine in the same mass content.

[0110] Example 8

[0111] One embodiment of the deep antioxidant EGCG nanocomposition of the present invention differs from Example 1 only in that the carrier amphiphilic polymer PGA is modified. 60 PMPC 120 The amount of [a specific ingredient] used was adjusted so that its mass percentage in the raw materials was 0.5%, and the amount of deionized water was adjusted so that the mass percentage of other components remained unchanged.

[0112] Example 9

[0113] One embodiment of the deep antioxidant EGCG nanocomposition of the present invention differs from Example 1 only in that the carrier amphiphilic polymer PGA is modified. 60 PMPC 120 The amount of [a specific ingredient] used was adjusted so that its mass percentage in the raw materials was 1%, and the amount of deionized water was adjusted so that the mass percentage of other components remained unchanged.

[0114] Example 10

[0115] One embodiment of the deep antioxidant EGCG nanocomposition of the present invention differs from Example 1 only in that the carrier amphiphilic polymer PGA is modified. 60 PMPC 120 The amount of [a specific ingredient] used was adjusted so that its mass percentage in the raw materials was 5%, and the amount of deionized water was adjusted so that the mass percentage of other components remained unchanged.

[0116] Example 11

[0117] This invention provides an embodiment of the deep antioxidant EGCG nanocomposition. The only difference between this embodiment and Example 1 is that the amount of the active substance EGCG is changed so that its mass percentage in the raw materials is 0.5%, and the amount of deionized water is adjusted so that the mass percentage of other components remains unchanged.

[0118] Example 12

[0119] This invention provides an embodiment of the deep antioxidant EGCG nanocomposition. The only difference between this embodiment and Example 1 is that the amount of the active substance EGCG is changed so that its mass percentage in the raw materials is 1%, and the amount of deionized water is adjusted so that the mass percentage of other components remains unchanged.

[0120] Example 13

[0121] This invention provides an embodiment of the deep antioxidant EGCG nanocomposition. The only difference between this embodiment and Example 1 is that the amount of the active substance EGCG is changed so that its mass percentage in the raw materials is 5%, and the amount of deionized water is adjusted so that the mass percentage of other components remains unchanged.

[0122] Comparative Example 1

[0123] The only difference between Comparative Example 1 and Example 1 is that the stabilizer phytosphingosine was not added, and the amount of deionized water was adjusted so that the mass percentage of other components remained unchanged.

[0124] Comparative Example 2

[0125] The only difference between Comparative Example 2 and Example 1 is that the amount of the active substance EGCG was changed so that its mass percentage in the raw materials was 7%, and the amount of deionized water was adjusted so that the mass percentage of other components remained unchanged.

[0126] Comparative Example 3

[0127] 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. Instead, the mixed solution obtained in step (2) is directly subjected to microfluidic high-pressure homogenization, with the pressure adjusted to 70 MPa, and the process is repeated 5 times to obtain the deep antioxidant nanocomposite.

[0128] Comparative Example 4

[0129] By weight percentage, 3% soybean lecithin, 0.1% cholesterol, and 3% EGCG were dissolved in ethanol at 30% of the weight of the finished product, and stirred at 400 rpm for 3 hours at 60°C to obtain a mixed solution.

[0130] The obtained mixed solution was evaporated by rotary evaporation (40℃, 250 rpm) to remove the ethanol solution. After the ethanol was completely removed, 93.9% of the product mass of deionized water was added, and the mixture was ultrasonically dispersed at 600W for 30 min. Finally, it was homogenized by microfluidic high pressure at 70 MPa for 5 times to obtain EGCG-encapsulated liposomes.

[0131] Comparative Example 5

[0132] By mass percentage, 3% EGCG and 15% caprylic / capric triglycerides were mixed and heated to 85°C until the EGCG was completely dissolved. Then, 10% butanediol by mass percentage was added to obtain the oil phase component.

[0133] The oil phase was added dropwise at a rate of 3 mL / min to 72% (w / w) deionized water. After the addition was complete, the mixture was stirred at 300 rpm for 15 min to obtain a mixed solution.

[0134] The mixed solution was dispersed by high-speed shearing at 10,000 rpm for 20 min; then it was homogenized by high-pressure microfluidic jet at a pressure of 80 MPa for 4 times to obtain EGCG nanoemulsion.

[0135] Comparative Example 6

[0136] The carrier used in Comparative Example 6 is the amphiphilic polymer PGA. 60 PMPC 120 Same as Example 1, with the same mass percentage of raw materials as in Example 1, and the preparation method of the nanocomposite is as follows:

[0137] (1) The prepared amphiphilic polymer PGA 60 PMPC 120 Stabilizer phytosphingosine and EGCG are added to 1,3-butanediol and heated to 60°C to obtain an alcoholic solution.

[0138] EGCG was added to deionized water and stirred at 400 rpm for 3 hours to obtain an aqueous solution.

[0139] (2) The alcohol phase solution above was added dropwise to the aqueous phase solution above. High-speed shearing was started simultaneously at 8000 rpm. The dropwise addition rate was 5 mL / min. After the dropwise addition was completed, the mixture was stirred at 400 rpm for 3 h to obtain a mixed solution.

[0140] (3) The dialysis and high-pressure homogenization operations in step (3) are the same as in Example 1, to obtain the nanocomposite.

[0141] Comparative Example 7

[0142] In Comparative Example 7, 3% by mass of EGCG was added to 97% by mass of deionized water and stirred at 300 rpm for 15 minutes until EGCG was completely dissolved, thus obtaining an EGCG aqueous solution.

[0143] Example 1

[0144] To investigate the encapsulation effect and storage stability of the deep antioxidant EGCG nanocomposition provided by this invention, the nanocompositions or other products in the above examples and comparative examples were tested as follows:

[0145] Encapsulation efficiency test:

[0146] Take 200 μL of the above-mentioned sample, centrifuge by ultrafiltration (9000 rpm, 30 min), take 5 μL of the filtrate and determine the content of EGCG in the filtrate and the content of free (unencapsulated) EGCG in the sample by high performance liquid chromatography (HPLC, Shimadzu, Japan).

[0147] Take another sample from the above test, add a mixed solution of methanol and deionized water, and ultrasonically demulsify 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, take 5 μL of the sample solution and determine the EGCG content using a high-performance liquid chromatograph (HPLC, Shimadzu, Japan). This yields the total EGCG content in the EGCG formulation. Calculate the encapsulation efficiency (EE) of the sample using the following formula:

[0148]

[0149] 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 with a mixture of methanol and deionized water.

[0150] Storage stability test:

[0151] The samples from the examples and comparative examples were left to stand at room temperature (25°C) for one month, two months, and three months, respectively. The encapsulation efficiency and EGCG content of the samples were then measured to test their storage stability. The results of the encapsulation efficiency and storage stability are shown in Table 1.

[0152] Table 1. Encapsulation efficiency and storage stability results of the examples and comparative examples.

[0153]

[0154]

[0155]

[0156]

[0157] As shown in Table 1:

[0158] 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 polymers used to prepare the nanocompositions all affect the storage stability of the product nanocompositions. In Examples 1-3, the degrees of polymerization of the hydrophobic block PGA were 60, 40, and 80, respectively. With the increase of the degree of polymerization of PGA, the change rate of EGCG content and encapsulation efficiency in the nanocompositions after 3 months of storage at room temperature first decreased and then increased. In Examples 1, 4, and 5, the degrees of polymerization of the hydrophilic block PMPC were 120, 80, and 160, respectively. With the increase of the degree of polymerization of PMPC, the change rate of EGCG content and encapsulation efficiency after 3 months of storage at room temperature also first decreased and then increased. In Example 1, when the degree of polymerization of PGA was 60 and the degree of polymerization of PMPC was 120, after 3 months of storage at room temperature, the change rates of EGCG content and encapsulation efficiency were -10.96% and -10.17%, respectively, which were the smallest changes, indicating the best storage stability of the product.

[0159] Comparing Examples 1, 6, and 7 with Comparative Example 1, it can be seen that the addition of stabilizers and their different types also affect storage stability.

[0160] In Example 1, the storage stability was optimal when the stabilizer was phytosphingosine and interacted with the other components.

[0161] Comparing Examples 1, 8-13, and Comparative Example 2, it is evident that the dosage of the active ingredient EGCG and the amphiphilic polymer affects the storage stability of the product. It can be seen that as the dosage of the amphiphilic polymer increases from 0.5-5%, or the dosage of EGCG increases from 0.5-7%, the rate of change in EGCG content and encapsulation efficiency of the deep antioxidant nanocomposite after 3 months of storage at room temperature initially decreases and then increases. In Example 1, the product exhibits optimal storage stability when both the dosage of the amphiphilic polymer and EGCG is 3%. Since the encapsulation efficiency and stability effects of the combined dosages of the active ingredient EGCG and the amphiphilic polymer used in Examples 11-13 and Comparative Example 2 are relatively poor, to conserve raw materials, further investigation into the other effects and performance of Examples 11-13 and Comparative Example 2 will not be conducted.

[0162] Comparing Example 1 and 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 impact on the stability of the nanocomposite; while compared with the use of liposomes (Comparative Example 4), nanoemulsion (Comparative Example 5) and a single aqueous solution dissolution method, the amphiphilic polymer encapsulation strategy of the present invention has significantly better storage stability.

[0163] Example 2

[0164] To investigate the transdermal absorption effect of the deep antioxidant EGCG nanocomposition provided by this invention, a transdermal absorption (penetration enhancement) experiment was used to evaluate the transdermal absorption (penetration enhancement) ability of the nanocompositions or other products in the examples and comparative examples. The specific methods are as follows:

[0165] In vitro transdermal experiments were conducted using a vertical diffusion cell, with nude mouse skin (abdominal skin, with subcutaneous fat and blood vessels removed) as the model. PBS solution was used as the receiving fluid. The skin patch was fixed between the supply and receiving cells, skin layer facing upwards, and equilibrated for 20 minutes. The nanocomposite samples from each example and comparative example were prepared into 10% (w / w) sample solutions, and these solutions were added to the supply cell. Receiving fluid was collected after 1 hour, 4 hours, 8 hours, and 24 hours. The receiving fluid was ultrasonically demulsified with a mixture of methanol and deionized water at a ratio of receiving fluid:methanol:water = 1:4:5 (V / V) for 30 minutes. After filtration through a 0.45 μm 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. Each experiment was performed in triplicate, and the arithmetic mean of the results was taken. The formula for calculating the cumulative permeation per unit area on the skin patch is as follows:

[0166] Among them, Q n The cumulative transmittance per unit area of ​​the sample at time t (μg / cm²) 2 ), A is the permeation area, C n C represents the concentration of the active ingredient measured at time t. i Table 2 shows the measured concentration of the active ingredient at time point t, where V is the total volume of the receiving solution and V0 is the sampling volume at each time point. The in vitro transdermal test results are shown in Table 2 below.

[0167] Table 2. In vitro transdermal test results of the examples and comparative examples.

[0168]

[0169] As shown in Table 2:

[0170] Comparing Examples 1-5, it can be seen that the degree of polymerization of the hydrophobic block PGA and the hydrophilic block PMPC in the amphiphilic polymers used to prepare the nanocompositions affects the transdermal permeability of the product nanocompositions. As the degree of polymerization of either the hydrophobic block PGA or the hydrophilic block PMPC in the amphiphilic polymer increases, the transdermal permeability of the nanocomposition first increases and then decreases. In Example 1, when the degree of polymerization of the hydrophobic block PGA in the amphiphilic polymer was 60 and the degree of polymerization of the hydrophilic block PMPC was 120, the cumulative permeation per unit area of ​​EGCG in the final product at 1h, 4h, 8h, and 24h was 6.42 μg / cm³. 2 21.47 μg / cm2 36.15 μg / cm 2 and 98.20 μg / cm 2 All of them are significantly higher than the nanocomposites of Examples 2-5, with the best transdermal penetration performance and more conducive to achieving deep anti-oxidation.

[0171] Comparing Examples 1, 6, and 7 with Comparative Example 1, it can be seen that the addition of stabilizers and their different types affect the transdermal permeability of the nanocomposition. In Example 1, when phytosphingosine was selected as the stabilizer and interacted with the other components, the transdermal permeability was optimal, which is beneficial for achieving deep antioxidant effects.

[0172] Comparing Examples 1 and 8-10, it can be seen that the amount of the active ingredient EGCG and the amphiphilic polymer affects the transdermal permeability of the nanocomposite. In Example 1, the amount of amphiphilic polymer was 3%, resulting in the best transdermal permeability.

[0173] Comparing Example 1 and 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 impact on the transdermal permeability of the nanocomposition; while compared with the use of liposomes (Comparative Example 4), nanoemulsion (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 effects.

[0174] Example 3

[0175] To investigate the cell affinity of the deep antioxidant nanocomposition provided by this invention, in vitro cytotoxicity experiments were used to evaluate the cell affinity of the nanocompositions or other products in the examples and comparative examples. The specific methods are as follows:

[0176] Using immortalized human keratinocytes (HaCaT cells) as human skin tissue model cells, the in vitro cytotoxicity of the samples was evaluated using the CCK-8 assay.

[0177] Logarithmic growth phase HaCaT cells (density 3000 cells / well) were seeded in 96-well plates and DMEM medium (containing 10% FBS and 1% P / S) was added. The HaCaT cells were incubated at 37°C in a cell culture incubator containing 5% CO2 for 24 h. The medium was then removed and replaced with a medium solution containing the test samples (concentrations set at 0.5%, 1.0%, 3.0%, and 5.0%), and the cells were incubated for another 48 h. CCK-8 reagent (10 μL / well) was added to the 96-well plates, and the plates were incubated for another 4 h. The absorbance of the solution in each well at 450 nm was measured using a microplate reader, and the viability of the HaCaT cells was calculated using the following formula:

[0178]

[0179] Among them, A 样品 Indicates the absorbance of the experimental group; A 对照 Indicates the absorbance of the control group; A 空白 The absorbance of the blank group is shown in Table 3 below. The results of the in vitro cytotoxicity test are shown in Table 3.

[0180] Table 3. In vitro cytotoxicity test results of the examples and comparative examples.

[0181]

[0182] As shown in Table 3:

[0183] 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 polymers used to prepare the nanocompositions both affect the cell affinity of the nanocompositions of the present invention. As the degree of polymerization of either the hydrophobic block PGA or 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 was 60 and that of PMPC was 120, after co-incubating the nanocomposition with HaCaT cells for 48 hours, the cell viability of samples with mass concentrations of 0.5%, 1.0%, 3.0%, and 5.0% were 104.2%, 103.3%, 101.4%, and 97.6%, respectively, demonstrating superior cell affinity. At a treatment concentration of 5%, the cell viability was significantly higher than that of the samples in Examples 2-5, indicating that Example 1 exhibits the best cell affinity and is more conducive to its efficacy at the cellular level.

[0184] Comparing Examples 1, 6, and 7 with Comparative Example 1, it can be seen that the addition and type of stabilizer affect the cell affinity of the nanocomposite. When phytosphingosine was chosen as the stabilizer, the cell survival rate was the highest and the cell affinity was the best, which is more conducive to its efficacy at the cellular level.

[0185] Comparing Examples 1 and 8-10, the cell affinity of the nanocomposite first increased and then decreased as the amount of amphiphilic polymer increased. When the amount of amphiphilic polymer added was 3%, the cell survival rate and cell affinity of the nanocomposite after co-incubation with HaCaT cells were the highest, which is more conducive to its efficacy at the cellular level.

[0186] Comparing Example 1 and 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 impact on the cell affinity of the nanocomposition (especially at higher concentrations); while compared with the use of liposomes (Comparative Example 4), nanoemulsion (Comparative Example 5) and a single aqueous solution dissolution method, the present invention adopts an amphiphilic polymer encapsulation strategy, which significantly reduces the cytotoxicity of the nanocomposition during its action and significantly improves the cell survival rate, which is conducive to exerting efficacy at the cellular level.

[0187] Example of effect 4

[0188] To preliminarily explore the antioxidant efficacy of the deep antioxidant nanocomposition provided by this invention, the antioxidant properties of the nanocompositions or other products in the examples and comparative examples were evaluated using a cellular reactive oxygen species (ROS) inhibition test. The specific methods are as follows:

[0189] The samples from the examples and comparative examples were prepared into 1% solutions by mass using PBS buffer (pH 6.8).

[0190] Human keratinocytes (HaCaT) were digested and prepared into a cell suspension, which was then seeded into 96-well plates at a density of 1.0 × 10⁶ cells per well. 5 -2.0×10 5 Cells were cultured in DMEM medium. After 24 hours, the medium was changed, and three groups were set up: a blank group (with PBS buffer, no light), a control group (with PBS buffer, under light), and an experimental group (containing the samples from the examples and comparative examples, respectively, at 10% of the cell culture medium volume). The cells were cultured for another 24 hours, and then the control and experimental groups were exposed to UVA light (6-10 J / cm²). 2 After the light exposure, 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 the cells were incubated in a CO2 incubator for 20 min. After incubation, the cells were washed three times with PBS, and serum-free DMEM culture medium was added again. ROS levels were detected using a fluorescence microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The relative ROS level was calculated as the ratio of the OD value of the experimental group (or control group) to the OD value of the blank group. The results are shown in Table 4.

[0191] Table 4. Relative ROS levels of HaCaT cells after light damage and sample treatment.

[0192] 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

[0193] As shown in Table 4:

[0194] The samples prepared by the deep antioxidant EGCG nanocompositions provided in Examples 1-10 of this invention showed that the relative ROS levels of HaCaT cells after light damage were lower than those of the control group. The nanocompositions prepared by the method of this invention have good antioxidant properties and can effectively remove reactive oxygen species in cells.

[0195] Furthermore, the samples prepared with the nanocompositions in Examples 1-10 showed significantly lower relative ROS levels in HaCaT cells after photodamage compared to Comparative Examples 1 and 3-6. This indicates that the nanocompositions in the examples within the scope of this invention have better antioxidant properties compared to other EGCG nanocomposition preparations or other encapsulation strategies.

[0196] A horizontal comparison of the relative ROS levels in the cells of each embodiment shows that the degree of polymerization of the hydrophobic block PGA and the hydrophilic block PMPC of the amphiphilic polymers used in the preparation steps, the amount of amphiphilic polymers used, and the addition and type of stabilizers all affect the antioxidant properties of the nanocompositions provided by this invention. This is related to the combined effects of the EGCG loading rate, stability, transdermal absorption performance, cell affinity, and the structure of the nanocomposition itself. Among them, the deep antioxidant nanocomposition prepared by the strategy used in Example 1 has the lowest relative ROS level in HaCaT cells after photodamage. After co-incubation with HaCaT cells after photodamage for 24 hours, the relative ROS level was 102.5%, which is close to the level of normal cells without photodamage. The nanocomposition prepared by the strategy in Example 1 has the best antioxidant properties.

[0197] Furthermore, it can be seen that compared with Comparative Example 7, the nanocomposition in Example 1 showed a significantly lower relative level of ROS in HaCaT cells after light damage. This indicates that compared with the unencapsulated EGCG monomer, the nanocomposition obtained by encapsulating the active ingredient EGCG with the amphiphilic polymer and specific stabilizer used in this invention can exert a better antioxidant effect.

[0198] Example 5

[0199] To further explore the deep antioxidant efficacy of the EGCG nanocomposition provided by this invention, the antioxidant signaling pathways of the nanocomposition of Example 1, which exhibits the best overall effect among the above-mentioned components, and other products in Comparative Examples 1 and 3-7 were evaluated experimentally. The specific methods are as follows:

[0200] When the stratum corneum of the skin is damaged, the skin barrier is compromised, leading to a hypermetabolic response. Cellular antioxidant mechanisms weaken, and the body's antioxidant system is impaired. Oxidative stress plays a dominant role in skin barrier repair, generating a large number of ROS free radicals that damage proteins, lipids, and DNA in skin tissue, thereby reducing cellular physiological activity, disrupting normal signaling pathways, and exacerbating inflammatory responses. H2O2, an oxidant that can freely diffuse between and within cells, can cause irreversible oxidative damage at excessively high concentrations and is often used in oxidative damage models.

[0201] HaCaT cells in the logarithmic growth phase were harvested at a rate of 2.5 × 10⁻⁶. 5 At a cell density of 5 cells / mL, 2 mL of the solution was seeded into each well of a 6-well plate, i.e., 5 × 10⁶ cells per well. 5 Cells were plated and cultured in a cell culture incubator for 12 hours. The original culture medium was discarded, and culture medium containing the sample (the mass percentage of each example and comparative example product in the sample was 0.5%) was added to pretreat the cells for 12 hours. After 12 hours, 100 μM H2O2 was added to each well, and the cells were incubated for 12 hours. After removing the culture medium, the cells were washed twice with fresh PBS solution.

[0202] Total RNA was extracted using the Simply P Total RNA Extraction Kit (Shanghai Beyotime Biotechnology Co., Ltd.) following the instructions. The purity of the obtained total RNA was then determined.

[0203] The total RNA extracted was reverse transcribed using the RT Master Mix for qPCR (gDNA digester plus, Shanghai Beyotime Biotechnology Co., Ltd.) kit to obtain cDNA.

[0204] Calculate the amount of RNA to be used based on the extracted RNA concentration, and add the following reagents sequentially 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, mix well by pipetting, and place in a PCR instrument. Incubate at 25℃ for 5 min, 42℃ for 40 min, and 85℃ for 2 min.

[0205] GAPDH was selected as the internal reference gene and synthesized according to the primer gene sequences in Table 5.

[0206] Prepare a 20 μL reaction system using NovoStart SYBR quantitative reagent and primers: 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).

[0207] According to the required amount for each group, mix the quantitative reagents and primers in advance, and mix the template with RNase-free water. Then, add them to the 96-well plate of the real-time PCR instrument in sequence. Make sure the real-time membrane is tightly attached to the 96-well plate to reduce the evaporation loss of each reagent during the reaction. Then, centrifuge at 3000 rpm for 3 min at 4℃ and run the instrument.

[0208] Table 5 Primer gene sequences

[0209]

[0210] The relative expression levels of the antioxidant NQO1, CAT, Nrf2, SOD1, and SOD2 genes in HaCaT cells pretreated with the nanocompositions or other products in Example 1 and Comparative Examples 1, 3-7 for oxidative damage repair are as follows: Figure 3 , 4 As shown in 5, 6, and 7.

[0211] Depend on Figure 3-7 As can be seen, the nanocomposition prepared by the preparation strategy in Example 1, after co-incubation with HaCaT cells damaged by H2O2, upregulated the NQO1, CAT, Nrf2, SOD1, and SOD2 genes in the cells by 5.12-fold, 9.67-fold, 5.83-fold, 11.4-fold, and 11.0-fold, respectively. In summary, the nanocomposition prepared by the preparation strategy in Example 1 of this invention has superior antioxidant properties. It can significantly mobilize the antioxidant defense mechanism in cells, initiate oxidative damage repair, and enhance the expression of antioxidant-related enzymes. Through good cell affinity and transdermal absorption and delivery, it can promote deep cellular antioxidant activity.

[0212] Meanwhile, 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 nanocomposites prepared by adjusting the preparation steps in comparative examples 3 and 6, the liposomes or nanoemulsions in comparative examples 4 and 5, and the unencapsulated single EGCG solution in comparative example 7, the present invention uses specific amphiphilic polymers and specific stabilizers to encapsulate the active ingredient EGCG. The resulting deep antioxidant EGCG nanocomposites can better achieve deep antioxidant performance under high encapsulation rate, stability, cell affinity, and transdermal absorption. By effectively regulating the expression of antioxidant-related genes, it achieves comprehensive and deeper antioxidant effects.

[0213] In summary, this invention improves the transdermal absorption and cell affinity of the nanocomposite by optimizing the amphiphilic polymer and selecting specific hydrophobic segments (PGA and PMPC) as carriers for the active ingredients. It achieves self-assembly of the nanocomposite with specific amounts of the amphiphilic polymer, phytosphingosine stabilizers, and the antioxidant active ingredient ECGG under specific preparation steps, thereby obtaining the nanocomposite and enhancing the loading capacity and stability of EGCG. Through optimized preparation steps, this invention prepares a deep antioxidant nanocomposite that significantly improves the transdermal absorption of EGCG, possesses high cell affinity, and provides deep antioxidant effects. The preparation strategy of this invention solves the problems of poor EGCG stability, poor transdermal absorption, and inability to penetrate skin cells to exert deep antioxidant effects in existing technologies, achieving deep antioxidant effects at the cellular level and possessing high application value.

[0214] 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 modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A deep-penetrating antioxidant epigallocatechin gallate nanocomposition, characterized in that, The raw materials for preparing the deep antioxidant epigallocatechin gallate nanocomposition include a carrier, stabilizer, active substance, water, and polyol; The active substance is epigallocatechin gallate; in the raw materials for preparing the deep antioxidant epigallocatechin gallate nanocomposition, the mass percentage of the amphiphilic polymer is 0.5-5%, the mass percentage of epigallocatechin gallate 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; The stabilizer is at least one of phytosphingosine, tetraacetylphytosphingosine, and salicylic acid phytosphingosine. The polyol is C2-C 10 Polyols; the support is an amphiphilic polymer, the general formula of which is X. m -Y n The X m It is polyglycolic acid, and the Y is... n It is poly-2-methacryloyloxyethyl phosphorylcholine; The degree of polymerization of the polyglycolic acid is m=40-80; The degree of polymerization of the poly(2-methacryloyloxyethylphosphorylcholine) is n=80-160; The preparation method of the amphiphilic polymer includes the following steps: S1, mix the initiator, glycolide and catalyst, and react at 80-150℃ for 6-24h to obtain polyglycolic acid; S2. Add the polyglycolic acid and the brominated reagent to the solvent and mix well. React at 0-25℃ for 12-36h to obtain brominated polyglycolic acid. S3. The brominated polyglycolic acid, 2-methacryloyloxyethyl phosphorylcholine, catalyst and catalyst ligand are added to the solvent and mixed. The mixture is reacted at 60-100°C for 24-72 h to obtain the amphiphilic polymer. The reactions in S1, S2 and S3 are all carried out under an inert atmosphere. 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. In step S1, the molar ratio of initiator, glycolide, and catalyst is initiator: glycolide: catalyst = 1:(20-40):(2-4). In step S2, the brominating agent is at least one of 2-bromoisobutyryl bromide, tert-butyl 2-bromoisobutyrate, and ethyl 2-bromoisovalerate. In step S2, the molar ratio of polyglycolic acid to brominated reagent is 1:(2-8). 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. In step S3, the molar ratio of brominated polyglycolic acid, 2-methacryloyloxyethyl phosphorylcholine, catalyst, and catalyst ligand is brominated polyglycolic acid: 2-methacryloyloxyethyl phosphorylcholine: catalyst: catalyst ligand = 1: (80-160): (0.8-1.2): (8-16).

2. The deep antioxidant epigallocatechin gallate nanocomposition as described in claim 1, characterized in that, The degree of polymerization of the polyglycolic acid is m=50-70, and the degree of polymerization of the poly(2-methacryloyloxyethylphosphorylcholine) is n=100-140. And / or, in the raw materials for preparing the deep antioxidant epigallocatechin gallate nanocomposition, the mass percentage of the amphiphilic polymer is 2-4%, the mass percentage of the epigallocatechin gallate 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.

3. The method for preparing the deep antioxidant epigallocatechin gallate nanocomposite as described in any one of claims 1-2, characterized in that, Includes the following steps: (1) The amphiphilic polymer, stabilizer, epigallocatechin gallate are heated and mixed in a polyol to obtain an alcohol phase solution; (2) The alcohol phase solution is added dropwise to water and subjected to high shear treatment to obtain a mixed solution; (3) Remove the polyol from the mixed solution and then homogenize it to obtain the deep antioxidant epigallocatechin gallate nanocomposition.

4. The preparation method of the deep antioxidant epigallocatechin gallate nanocomposite as described in claim 3, characterized in that, In step (1), the temperature for heating and mixing is 35-80℃; And / or, in 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 continues until the alcohol phase solution is added; And / or, in step (3), dialysis is used to remove polyols, and the dialysis bag used for dialysis has a pore size of 300-1000 Da; And / or, the homogenization pressure in step (3) is 50-120 MPa, and the number of homogenization cycles is 2-8.

5. Use of the deep antioxidant epigallocatechin gallate nanocomposition as described in any one of claims 1-2 in antioxidant cosmetics.

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