An EGCG preparation with ultra-high stability and high-efficiency anti-aging effect, and its preparation method and application

Abstract

An EGCG preparation with ultra-high stability and high-efficiency anti-aging effect, a preparation method and application thereof, wherein EGCG is first encapsulated by a polymer wall material, and then an esterification reaction is carried out with a cross-linking agent containing two boric acid groups to obtain the EGCG preparation; the mass ratio of the polymer wall material to the cross-linking agent is 1:0.5-2.0; the polymer wall material has amphiphilicity, and is composed of a polymer monomer e Caprolactone is obtained by ring-opening polymerization to obtain polycaprolactone, which is then obtained by bromination reaction to obtain brominated polycaprolactone, and then obtained by atom transfer radical polymerization of the polymerization monomer poly(ethylene glycol) methacrylate. The present invention generates borate bonds through cross-linking reaction, and its unique network structure firmly wraps EGCG, making it more stable; in addition, when in a high-active oxygen ROS environment, EGCG can be efficiently released, thereby achieving a high-efficiency antioxidant effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics and external skin medicines, and in particular relates to an EGCG preparation with ultra-high stability and high-efficiency anti-aging effect, and a preparation method and application thereof. Background Art

[0002] Long-term exposure to sunlight can easily cause aging of human skin. The damage caused by sunlight to human skin mainly comes from ultraviolet rays, which are divided into UVA (320~400nm) and UVB (280~320nm). Ultraviolet rays not only cause skin aging, but also trigger p53 inhibitory gene mutations in skin cells, activate the production of reactive oxygen species ROS, and thus damage DNA. For a long time, the focus of attention on the aging of human skin caused by sunlight exposure has been on protecting the skin from ultraviolet rays. The most common method is to apply sunscreen, relying on various broad-spectrum sunscreens to absorb and / or block ultraviolet rays in sunlight from damaging the skin, such as physical sunscreen ingredients such as titanium dioxide and zinc oxide; or chemical sunscreen ingredients such as octyl methoxycinnamate and oxybenzone. Although these ingredients can effectively block ultraviolet rays, long-term use of these chemicals may react with molecules in the skin, causing irritation, photosensitivity, and even dermatitis. In addition, the sunscreens in the prior art cannot completely block ultraviolet rays. Under long-term exposure to sunlight, the skin will still be damaged to a certain extent.

[0003] Tea polyphenols is a general term for polyphenol compounds in tea. Catechins, as the main components of tea polyphenols, include epigallocatechin gallate (EGC), epicatechin (EC), epigallocatechin gallate (EGCG), epicatechin gallate (ECG), etc. Among them, EGCG is the component with the highest content in catechins, accounting for 50-60%. Studies have shown that it has significant antioxidant effects, can effectively remove active oxygen free radicals, protect cells from damage, and thus delay skin aging. However, in actual applications, EGCG has problems such as poor stability and easy color change. Especially when it is actually used in cosmetics, its transdermal absorption is extremely low and it cannot effectively penetrate the stratum corneum of the skin to exert its effect.

[0004] In order to improve the above-mentioned problems of EGCG in application, liposomes or alcohol bodies are often used in the prior art to encapsulate EGCG. The Chinese invention patent application with publication number CN107049952A discloses a preparation method and application of catechin liposomes prepared by a multiple emulsion dispersion method, but the method uses chloroform, a controlled drug that is easy to make drugs, as a dissolution, which has certain toxicity and its encapsulation rate is not high (<80%); the Chinese invention patent application with publication number CN118717542A discloses a preparation method and application of non-toxic and safe tea polyphenol liposomes, and its encapsulation rate can reach 96.15%, but its stability inspection time is only 24h, which fails to effectively explain the stability of the product. In addition, in the prior art, most of the EGCG encapsulated products enable EGCG to achieve a sustained release effect and continue to exert its efficacy in the skin. However, the skin after sunlight exposure is under ultraviolet radiation stress, and the active oxygen ROS in the skin cells increases significantly, causing damage to the cell DNA. Therefore, how to efficiently achieve skin anti-oxidation and anti-aging is also a technical problem that needs to be solved. Summary of the invention

[0005] In order to overcome the problems existing in the practical application of EGCG in the prior art, the primary purpose of the present invention is to provide a method for preparing an EGCG preparation with ultra-high stability and high anti-aging efficacy.

[0006] Another object of the present invention is to provide an EGCG preparation prepared by the above method with ultra-high stability and high anti-aging efficacy.

[0007] The present invention also provides the application of the EGCG preparation prepared by the above method with ultra-high stability and high-efficiency anti-aging effect.

[0008] The purpose of the present invention is achieved through the following solutions:

[0009] A method for preparing an EGCG preparation with ultra-high stability and high-efficiency anti-aging effect, comprising first encapsulating EGCG with a polymer wall material, and then undergoing an esterification reaction with a cross-linking agent containing two boric acid groups to obtain the EGCG preparation; wherein the mass ratio of the polymer wall material to the cross-linking agent is 1:0.5-2.0; the cross-linking agent contains two boric acid groups and can undergo an esterification cross-linking reaction with hydroxyl groups on the polymer wall material.

[0010] The polymer wall material has amphiphilicity and is composed of polymerized monomers. e -caprolactone was obtained by ring-opening polymerization (ROP) to obtain polycaprolactone (PCL), which was then brominated to obtain brominated polycaprolactone, which was then obtained by atom transfer radical polymerization (ATRP) from the polymerization monomer poly(ethylene glycol) methacrylate.

[0011] Preferably, the steps include:

[0012] (S1) adding the polymer wall material and EGCG to the polyol, heating and stirring to obtain a phase A solution;

[0013] (S2) adding the phase A solution dropwise into water, and after the addition is completed, continuing to stir to obtain a mixed solution, removing the polyol in the mixed solution and then adding a crosslinking agent, stirring sufficiently to obtain a crosslinked polymer mixed solution;

[0014] (S3) The cross-linked polymer mixed solution is subjected to nano-dispersion treatment, and then freeze-dried to obtain an EGCG preparation.

[0015] Preferably, the mass ratio of the polymer wall material to the cross-linking agent is 1:1±0.5; the mass ratio of the polymer wall material to EGCG is 1:0.2-1.0.

[0016] Preferably, the mass ratio of the polymer wall material to EGCG in step (S1) is 1:0.4±0.1; the mass ratio of the polymer wall material to the polyol is 1:2-10; the polyol is at least one of 1,3-propylene glycol, glycerol, 1,4-butanediol, pentanediol, 1,2-hexanediol, 1,6-hexanediol, octyldodecanol, ethylene glycol, dipropylene glycol, and ethoxydiglycol.

[0017] Preferably, the cross-linking agent described in step (S2) is one or more of 1,4-phenylenediboronic acid, 1,3-phenylenediboronic acid, 2,6-naphthylideneboronic acid, anthracene-9,10-diboric acid, 4,4'-biphenylenediboric acid, and B,B'-[1,1':4',1''-triphenyl]-4,4''-diylbis[boric acid].

[0018] Preferably, the heating temperature in step (S1) is 30-60°C, the stirring speed is 100-500 rpm, and the stirring time is 30-90 min.

[0019] Preferably, the dropping speed in step (S2) is 1-10 mL / min; the stirring speed is 100-400 rpm; the stirring time is 10-30 min; the cross-linked polymer mixed solution is dialyzed with water; the polyol in the mixed solution is selectively dialyzed with water, and the dialysis conditions are: the molecular weight cutoff of the dialysis bag used is 500-5000Da; the dialysis time is 12-48h; and the water is replaced every 2-4h.

[0020] In the present invention, the purpose of using two dialysis treatments is that, firstly, the first dialysis operation is to remove the polyol solvent in step (S1), which is beneficial for the polymer wall material to better encapsulate the active ingredient EGCG, and to avoid the cross-linking agent added in the subsequent step from reacting with the polyol solvent, resulting in the loss of the cross-linking agent, and failing to achieve the ideal cross-linking effect of the polymer wall material. Therefore, the esterification reaction system does not contain alcohol solvents; secondly, the second dialysis operation is to remove the unreacted cross-linking agent, so that the prepared EGCG preparation is more stable.

[0021] Preferably, the nano-dispersion treatment described in step (S3) includes high-speed shear dispersion treatment and / or microfluidization homogenization treatment, wherein the high-speed shear dispersion treatment is performed by using a high-speed shear homogenizer for shear homogenization, the rotation speed is 6000-12000 rpm, and the shear dispersion time is 10-20 min; the microfluidization homogenization treatment is performed by using a microfluidizer for homogenization, the homogenization pressure is 50-120 MPa, and the number of homogenization cycles is 1-8 times.

[0022] Preferably, the molecular weight of the polymer wall material is n =32000-63000; the degree of polymerization of the polycaprolactone is 40±20.

[0023] Preferably, the method for preparing the polymer wall material comprises the following steps:

[0024] (1) e -caprolactone is added to a polyol initiator with hydroxyl groups at both ends, and a tin catalyst is added to the reaction system under the protection of an inert gas, and the reaction is carried out at 80-160°C for 8-18 hours to prepare polycaprolactone;

[0025] (2) dissolving the polycaprolactone in an organic solvent, adding 2-bromoisobutyryl bromide under the protection of an inert gas, reacting at -5-5°C for 2-4 hours, and then continuing to react at 25±15°C for 12-48 hours to obtain brominated polycaprolactone;

[0026] (3) Dissolving the polymerization monomers poly(ethylene glycol) methacrylate, brominated polycaprolactone, and N,N,N',N',N''-pentamethyldiethylenetriamine in an organic solvent, adding a copper salt catalyst to the reaction system under the protection of an inert gas, and reacting at 40-80° C. for 12-48 hours to prepare the polymer wall material.

[0027] Preferably, in step (1), the polyol (initiator) is terminated with hydroxyl groups at both ends, in order to require a sufficiently small steric hindrance to allow the polymerization monomers to e-Caprolactone undergoes ROP reaction at both ends of the initiator. If the two reaction sites (hydroxyl groups) are adjacent, the steric hindrance of the polymerization monomer reaction is too large, which is not conducive to the reaction; the polyol with hydroxyl groups at both ends is at least one of 1,4-butanediol, 1,5-pentanediol, and 1,6-hexanediol.

[0028] Preferably, the tin catalyst is at least one of stannous isooctanoate and stannous octoate; e The molar ratio of -caprolactone to the catalyst is 1:0.005-0.02; the polyol initiator having hydroxyl groups at both ends and e -The molar ratio of caprolactone is 1:10-40.

[0029] Preferably, the molar ratio of polycaprolactone to 2-bromoisobutyryl bromide in step (2) is 1:4.0-12.0.

[0030] Preferably, the organic solvent in steps (2) and (3) includes at least one of tetrahydrofuran, chloroform, N,N-dimethylformamide and dimethyl sulfoxide.

[0031] Preferably, in step (3), the molar ratio of the brominated polycaprolactone to the polymerizable monomers poly(ethylene glycol) methacrylate, N,N,N',N',N''-pentamethyldiethylenetriamine and the copper salt catalyst is 1:50-150:5-15:0.5-5; and the copper salt catalyst is at least one of CuBr, CuCl and CuI.

[0032] After the reaction in step (1) is completed, a purification step is further included: slowly dropping the reaction mixture into a precipitant, collecting the solid product after precipitation purification, and drying it at 40-60° C. for 12-48 hours to obtain the polycaprolactone. Preferably, in the precipitation purification process, the precipitant includes at least one of n-hexane, ether, and petroleum ether.

[0033] After the reaction in step (2) is completed, a purification step is further included: the reaction mixture is slowly added dropwise to a precipitant, and after purification by precipitation, a solid product is collected and dried at 40-60° C. for 12-48 hours to obtain the brominated polycaprolactone.

[0034] After the reaction in step (3) is completed, a purification step is further included: the reaction mixture is transferred to a dialysis bag and dialyzed in deionized water for 48-96 hours to remove unreacted polymer monomer poly(ethylene glycol) methacrylate and catalyst, and the deionized water is replaced every 4 hours. After the dialysis is completed, the liquid in the dialysis bag is freeze-dried for 12-48 hours to obtain a refined polymer wall material. Preferably, the molecular weight cutoff of the dialysis bag is 500-4500 Da.

[0035] The inert gas used in steps (1), (2) and (3) is at least one of nitrogen, helium and argon.

[0036] The stirring method in steps (S1) and (S2) is mechanical stirring or magnetic stirring.

[0037] The freeze drying in step (S3) has a drying time of 24-72 hours.

[0038] The EGCG preparation with ultra-high stability and high anti-aging efficacy prepared by the above method is used in cosmetics or in the preparation of external skin medicines.

[0039] The polymer wall material prepared by the present invention is used to encapsulate the active ingredient EGCG to obtain an EGCG preparation with ultra-high stability and high-efficiency anti-aging effect. In the present invention, the boric acid group in the cross-linking agent can undergo an esterification reaction with the hydroxyl group on the side chain of the polymer wall material polymethacrylic acid poly(ethylene glycol) to generate a borate ester bond, form a cross-link between molecular chains, and form a network polymer structure, which can better encapsulate the active ingredient EGCG, protect EGCG from sudden release, and improve the stability of the EGCG preparation. In addition, in an environment with high active oxygen (the active oxygen content in aged skin damaged by sun exposure increases), the high ROS content can stimulate the response to the breakage of the borate ester bond, the network structure is destroyed, and EGCG can be efficiently released, reducing the active oxygen content.

[0040] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0041] (1) Based on the prepared polymer wall material, the present invention adopts a molecular chain cross-linking reaction method to effectively improve the polymer wall material's ability to encapsulate the active substance EGCG. The cross-linking between the molecular chains can form a unique network structure, which has better stability than the EGCG preparation that has not been cross-linked. It prevents the EGCG preparation from being suddenly released during the storage process or application process, and can effectively maintain the bioactivity / bioavailability of the EGCG preparation.

[0042] (2) The cross-linking agent in the present invention undergoes a cross-linking reaction between the molecular chains and the hydroxyl groups on the side chains of the polymer wall material to generate borate bonds, and its unique network structure firmly wraps the EGCG. When in a high-active oxygen ROS environment, the high ROS content in the skin cells can stimulate the response to the breakage of the borate bonds. At this time, the network structure in the EGCG preparation is destroyed, and EGCG can be efficiently released, thereby achieving efficient anti-oxidation. It can reduce the active oxygen level in cells damaged by ultraviolet irradiation to the level of normal cells in a short time (2h), and has a very efficient antioxidant effect.

[0043] (3) Compared with EGCG preparations that have not been cross-linked and modified, the EGCG preparations of the present invention can also repair the DNA of cells damaged by ultraviolet irradiation in a short time (2 hours), promote the production of ATP in cells, and efficiently restore cells to normal cell levels, thus having a more efficient anti-aging effect than EGCG preparations that have not been cross-linked and modified.

[0044] (4) Compared with free EGCG (without encapsulation and / or cross-linking modification), liposome formulations and ethosome formulations, the EGCG preparation of the present invention exhibits higher stability and more efficient antioxidant and anti-aging effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 For the present invention c -H2AX method DNA damage repair results.

[0046] Figure 2 This is a graph showing the results of an experiment on ATP generating ability of the present invention. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. If the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not indicated for the reagents or instruments used, they are all conventional products that can be purchased commercially. The reagents used in the examples can be purchased conventionally from the market unless otherwise specified.

[0048] In the embodiments of the present invention, EGCG (98%), e -Caprolactone (≥99%), poly(ethylene glycol) methacrylate (Mn=500), methoxypoly(ethylene glycol) acrylate (Mn=480), 1,4-phenylenediboronic acid (≥97%), and 4,4'-biphenylenediboronic acid (≥95%) can be purchased directly from Shanghai Aladdin Biotechnology Co., Ltd.; 2,6-dinaphthylboronic acid (≥97%) can be purchased directly from Shanghai MacLean Biochemical Technology Co., Ltd.; anthracene-9,10-diboric acid (98%) and B,B'-[1,1':4',1''-triphenyl]-4,4''-diylbis[boric acid] (98%, CAS No.: 204923-11-5) can be purchased directly from Zhengzhou Alpha Chemical Co., Ltd.

[0049] Example 1

[0050] (1) Polymer PPEGMA 40 - b -PCL 20 - b -PPEGMA 40 Preparation (taking the preparation of 1.0mmol as an example): e -Caprolactone (M n =114.14, 2.28 g, 20.0 mmol) was added to 1,4-butanediol (M n =90.12, 0.09 g, 1.0 mmol), under nitrogen protection, the catalyst stannous octoate (M n =405.10, 0.08 g, 0.2 mmol), reacted at 130 °C for 12 h, the reaction mixture was slowly added dropwise to n-hexane, and after precipitation and purification, the solid product was collected and dried at 50 °C for 24 h to obtain PCL 20 .

[0051] PCL 20 (2.37 g, 1.0 mmol) was dissolved in tetrahydrofuran (100 mL) and 2-bromoisobutyryl bromide (M n =229.9, 2.30 g, 10.0 mmol), reacted at 0 ° C for 4 h, then heated to room temperature and continued to react for 24 h, the reaction mixture was slowly added dropwise to n-hexane, and after precipitation purification, the solid product was collected and dried at 50 ° C for 24 h to obtain Br-PCL 20 -Br.

[0052] Br-PCL 20 -Br (2.67 g, 1.0 mmol), poly(ethylene glycol) methacrylate (M n =500, 40.00 g, 80.0 mmol), N,N,N',N',N''-pentamethyldiethylenetriamine (M n =173.30, 1.73 g, 10.0 mmol) was dissolved in tetrahydrofuran (100 mL) and the catalyst CuBr (M n =143.45, 0.29 g, 2.0 mmol), reacted at 60°C for 24 h, transferred the reaction mixture to a dialysis bag (molecular weight cutoff of 3000 Da), and dialyzed in deionized water (1 L) for 72 h, with the deionized water (1 L) replaced every 4 hours. After the dialysis, the liquid in the dialysis bag was freeze-dried for 24 h to obtain the polymer wall material PPEGMA 40 - b -PCL 20 - b -PPEGMA 40 (Number average molecular weight M n =42373).

[0053] (2) 5 g of the prepared polymer wall material PPEGMA 40 - b-PCL 20 - b -PPEGMA 40 2 g of EGCG and 2 g of EGCG were added to 20 g of 1,2-hexanediol, heated to 50 °C, and stirred (at a speed of 300 rpm) for 60 min to obtain a phase A solution; the phase A solution was slowly added dropwise (dropping speed: 5 mL / min) to 68 g of deionized water. After the addition was completed, stirring was continued (at a speed of 300 rpm) for 20 min; the mixed solution was transferred to a dialysis bag (molecular weight cutoff of 3000 Da), dialyzed with deionized water (2 L) for 24 h, and the deionized water (2 L) was replaced every 4 hours; after the dialysis was completed, 5 g of the cross-linking agent 1,4-phenylenediboric acid was added, stirred (450 rpm) for 4 h; the above solution was transferred to a dialysis bag (molecular weight cutoff of 3000 Da) again, and dialyzed with deionized water (2 L) for 48 h, and the deionized water (2 L) was replaced every 4 hours; after the dialysis was completed, the solution in the dialysis bag was treated with high-pressure microfluidization homogenization at a homogenization pressure of 80 MPa and four times; after freeze-drying for 48 h, the EGCG preparation was obtained.

[0054] Example 2-5 (different types of cross-linking agents)

[0055] Example 2

[0056] The EGCG preparation was prepared by replacing 5 g of the cross-linking agent 1,4-phenyldiboric acid in Example 1 with 5 g of 4,4'-biphenyldiboric acid, while keeping other conditions unchanged.

[0057] Example 3

[0058] The EGCG preparation was prepared by replacing 5 g of the cross-linking agent 1,4-phenylenediboric acid in Example 1 with 5 g of 2,6-naphthylideneboric acid, while keeping other conditions unchanged.

[0059] Example 4

[0060] The EGCG preparation was prepared by replacing 5 g of the cross-linking agent 1,4-phenyldiboric acid in Example 1 with 5 g of anthracene-9,10-diboric acid, while keeping other conditions unchanged.

[0061] Example 5

[0062] The EGCG preparation was prepared by replacing 5 g of the cross-linking agent 1,4-phenylenediboronic acid in Example 1 with 5 g of B,B′-[1,1′:4′,1′′-triphenyl]-4,4′′-diylbis[boric acid] while keeping other conditions unchanged.

[0063] Example 6-8 (different amounts of cross-linking agent added)

[0064] Example 6 (polymer: cross-linking agent = 1:0.5)

[0065] The EGCG preparation was prepared by replacing 5 g of the cross-linking agent 1,4-phenylenediboric acid in Example 1 with 2.5 g of 1,4-phenylenediboric acid, and replacing 68 g of deionized water with 70.5 g of deionized water, while keeping the other conditions unchanged.

[0066] Example 7 (polymer: cross-linking agent = 1:1.5)

[0067] The EGCG preparation was prepared by replacing 5 g of the cross-linking agent 1,4-phenylenediboric acid in Example 1 with 7.5 g of 1,4-phenylenediboric acid, and replacing 68 g of deionized water with 65.5 g of deionized water, while keeping the other conditions unchanged.

[0068] Example 8 (polymer: cross-linking agent = 1:2)

[0069] The EGCG preparation was prepared by replacing 5 g of the cross-linking agent 1,4-phenylenediboric acid in Example 1 with 10 g of 1,4-phenylenediboric acid, and replacing 68 g of deionized water with 63 g of deionized water, while keeping the other conditions unchanged.

[0070] Examples 9-10 (different degrees of polymerization of PCL)

[0071] Example 9

[0072] (1) Polymer PPEGMA 40 - b -PCL 10 - b -PPEGMA 40 Preparation (taking the preparation of 1.0mmol as an example): e -Caprolactone (M n =114.14, 1.14 g, 10.0 mmol) was added to 1,4-butanediol (M n =90.12, 0.09 g, 1.0 mmol), under nitrogen protection, the catalyst stannous octoate (M n =405.10, 0.04 g, 0.1 mmol), reacted at 130 °C for 12 h, the reaction mixture was slowly added dropwise to n-hexane, and after precipitation and purification, the solid product was collected and dried at 50 °C for 24 h to obtain PCL 10 .

[0073] PCL 10 (1.23 g, 1.0 mmol) was dissolved in tetrahydrofuran (100 mL) and 2-bromoisobutyryl bromide (M n=229.9, 2.30 g, 10.0 mmol), reacted at 0 ° C for 4 h, then heated to room temperature and continued to react for 24 h, the reaction mixture was slowly added dropwise to n-hexane, and after precipitation purification, the solid product was collected and dried at 50 ° C for 24 h to obtain Br-PCL 10 -Br.

[0074] Br-PCL 10 -Br (1.53 g, 1.0 mmol), poly(ethylene glycol) methacrylate (M n =500, 40.00 g, 80.0 mmol), N,N,N',N',N''-pentamethyldiethylenetriamine (M n =173.30, 1.73 g, 10.0 mmol) was dissolved in tetrahydrofuran (100 mL) and the catalyst CuBr (M n =143.45, 0.29 g, 2.0 mmol), reacted at 60°C for 24 h, transferred the reaction mixture to a dialysis bag (molecular weight cutoff of 3000 Da), and dialyzed in deionized water (1 L) for 72 h, with the deionized water (1 L) replaced every 4 hours. After the dialysis, the liquid in the dialysis bag was freeze-dried for 24 h to obtain the polymer wall material PPEGMA 40 - b -PCL 10 - b -PPEGMA 40 (Number average molecular weight M n =41232).

[0075] (2) 5 g of the polymer wall material PPEGMA in Example 1 40 - b -PCL 20 - b -PPEGMA 40 and replaced with a polymer wall material PPEGMA with a mass of 5g 40 - b -PCL 10 - b -PPEGMA 40 , and the other conditions remained unchanged to prepare the EGCG preparation.

[0076] Example 10

[0077] (1) Polymer PPEGMA 40 - b -PCL 30 - b -PPEGMA 40 Preparation (taking the preparation of 1.0mmol as an example): e -Caprolactone (M n =114.14, 3.42 g, 30.0 mmol) was added to 1,4-butanediol (M n =90.12, 0.09 g, 1.0 mmol), under nitrogen protection, the catalyst stannous octoate (M n =405.10, 0.04 g, 0.3 mmol), reacted at 130 °C for 12 h, the reaction mixture was slowly added dropwise to n-hexane, and after precipitation and purification, the solid product was collected and dried at 50 °C for 24 h to obtain PCL 30 .

[0078] PCL 30 (3.51 g, 1.0 mmol) was dissolved in tetrahydrofuran (100 mL) and 2-bromoisobutyryl bromide (M n =229.9, 2.30 g, 10.0 mmol), reacted at 0 ° C for 4 h, then heated to room temperature and continued to react for 24 h, the reaction mixture was slowly added dropwise to n-hexane, and after precipitation purification, the solid product was collected and dried at 50 ° C for 24 h to obtain Br-PCL 30 -Br.

[0079] Br-PCL 30 -Br (3.81 g, 1.0 mmol), poly(ethylene glycol) methacrylate (M n =500, 40.00 g, 80.0 mmol), N,N,N',N',N''-pentamethyldiethylenetriamine (M n =173.30, 1.73 g, 10.0 mmol) was dissolved in tetrahydrofuran (100 mL) and the catalyst CuBr (M n =143.45, 0.29 g, 2.0 mmol), reacted at 60°C for 24 h, transferred the reaction mixture to a dialysis bag (molecular weight cutoff of 3000 Da), and dialyzed in deionized water (1 L) for 72 h, with the deionized water (1 L) replaced every 4 hours. After the dialysis, the liquid in the dialysis bag was freeze-dried for 24 h to obtain the polymer wall material PPEGMA 40 - b -PCL 30 - b -PPEGMA 40 (Number average molecular weight M n =43514).

[0080] (2) 5 g of the polymer wall material PPEGMA in Example 1 40 - b-PCL 20 - b -PPEGMA 40 and replaced with a polymer wall material PPEGMA with a mass of 5g 40 - b -PCL 30 - b -PPEGMA 40 , and the other conditions remained unchanged to prepare the EGCG preparation.

[0081] Examples 11-13 (Different degrees of polymerization of PPEGMA)

[0082] Embodiment 11

[0083] (1) Polymer PPEGMA 30 - b -PCL 20 - b -PPEGMA 30 Preparation (taking the preparation of 1.0mmol as an example): Br-PCL 20 The preparation steps of Br-PCL are the same as those in Example 1. 20 -Br (2.67 g, 1.0 mmol), poly(ethylene glycol) methacrylate (M n =500, 30.00 g, 60.0 mmol), N,N,N',N',N''-pentamethyldiethylenetriamine (M n =173.30, 1.73 g, 10.0 mmol) was dissolved in tetrahydrofuran (100 mL) and the catalyst CuBr (M n =143.45, 0.29 g, 2.0 mmol), reacted at 60°C for 24 h, transferred the reaction mixture to a dialysis bag (molecular weight cutoff of 3000 Da), and dialyzed in deionized water (1 L) for 72 h, with the deionized water (1 L) replaced every 4 hours. After the dialysis, the liquid in the dialysis bag was freeze-dried for 24 h to obtain the polymer wall material PPEGMA 30 - b -PCL 20 - b -PPEGMA 30 (Number average molecular weight M n =32373).

[0084] (2) 5 g of the polymer wall material PPEGMA in Example 1 40 - b -PCL 20 - b -PPEGMA 40 and replaced with a polymer wall material PPEGMA with a mass of 5g30 - b -PCL 20 - b -PPEGMA 30 , and the other conditions remained unchanged to prepare the EGCG preparation.

[0085] Example 12

[0086] (1) Polymer PPEGMA 50 - b -PCL 20 - b -PPEGMA 50 Preparation (taking the preparation of 1.0mmol as an example): Br-PCL 20 The preparation steps of Br-PCL are the same as those in Example 1. 20 -Br (2.67 g, 1.0 mmol), poly(ethylene glycol) methacrylate (M n =500, 50.00 g, 100.0 mmol), N,N,N',N',N''-pentamethyldiethylenetriamine (M n =173.30, 1.73 g, 10.0 mmol) was dissolved in tetrahydrofuran (100 mL) and the catalyst CuBr (M n =143.45, 0.29 g, 2.0 mmol), reacted at 60°C for 24 h, transferred the reaction mixture to a dialysis bag (molecular weight cutoff of 3000 Da), and dialyzed in deionized water (1 L) for 72 h, with the deionized water (1 L) replaced every 4 hours. After the dialysis, the liquid in the dialysis bag was freeze-dried for 24 h to obtain the polymer wall material PPEGMA 50 - b -PCL 20 - b -PPEGMA 50 (Number average molecular weight M n =52373).

[0087] (2) 5 g of the polymer wall material PPEGMA in Example 1 40 - b -PCL 20 - b -PPEGMA 40 and replaced with a polymer wall material PPEGMA with a mass of 5g 50 - b -PCL 20 - b -PPEGMA 50 , and the other conditions remained unchanged to prepare the EGCG preparation.

[0088] Embodiment 13

[0089] (1) Polymer PPEGMA 60 - b -PCL 20 - b -PPEGMA 60 Preparation (taking the preparation of 1.0mmol as an example): Br-PCL 20 The preparation steps of Br-PCL are the same as those in Example 1. 20 -Br (2.67 g, 1.0 mmol), poly(ethylene glycol) methacrylate (M n =500, 60.00 g, 120.0 mmol), N,N,N',N',N''-pentamethyldiethylenetriamine (M n =173.30, 1.73 g, 10.0 mmol) was dissolved in tetrahydrofuran (100 mL) and the catalyst CuBr (M n =143.45, 0.29 g, 2.0 mmol), reacted at 60°C for 24 h, transferred the reaction mixture to a dialysis bag (molecular weight cutoff of 3000 Da), and dialyzed in deionized water (1 L) for 72 h, with the deionized water (1 L) replaced every 4 hours. After the dialysis, the liquid in the dialysis bag was freeze-dried for 24 h to obtain the polymer wall material PPEGMA 60 - b -PCL 20 - b -PPEGMA 60 (Number average molecular weight M n =62373).

[0090] (2) 5 g of the polymer wall material PPEGMA in Example 1 40 - b -PCL 20 - b -PPEGMA 40 and replaced with a polymer wall material PPEGMA with a mass of 5g 60 - b -PCL 20 - b -PPEGMA 60 , and the other conditions remained unchanged to prepare the EGCG preparation.

[0091] Examples 14-17 (Different feed ratios of polymer and polyphenol)

[0092] Example 14 (polymer: EGCG = 1:0.2)

[0093] The EGCG preparation was prepared by replacing 2 g of EGCG in Example 1 with 1 g of EGCG, and replacing 68 g of deionized water with 69 g of deionized water, while keeping other conditions unchanged.

[0094] Example 15 (polymer:EGCG=1:0.6)

[0095] The EGCG preparation was prepared by replacing 2 g of EGCG in Example 1 with 3 g of EGCG and replacing 68 g of deionized water with 67 g of deionized water while keeping other conditions unchanged.

[0096] Example 16 (polymer: EGCG = 1:0.8)

[0097] The EGCG preparation was prepared by replacing 2 g of EGCG in Example 1 with 4 g of EGCG and replacing 68 g of deionized water with 66 g of deionized water while keeping other conditions unchanged.

[0098] Example 17 (polymer: EGCG = 1:1)

[0099] The EGCG preparation was prepared by replacing 2 g of EGCG in Example 1 with 5 g of EGCG, and replacing 68 g of deionized water with 65 g of deionized water, while keeping other conditions unchanged.

[0100] Example 18-19 (different process parameters)

[0101] Embodiment 18

[0102] Polymer wall material PPEGMA 40 - b -PCL 20 - b -PPEGMA 40 The preparation steps are the same as in Example 1.

[0103] The prepared polymer wall material PPEGMA with a mass of 5 g 40 - b -PCL 20 - b -PPEGMA 402 g of EGCG and 2 g of EGCG were added to 40 g of glycerol, heated to 30 °C, and stirred (speed: 500 rpm) for 80 min to obtain phase A solution; the phase A solution was slowly added dropwise (dropping speed: 8 mL / min) to 48 g of deionized water. After the addition was completed, stirring was continued (speed: 300 rpm) for 20 min; the mixed solution was transferred to a dialysis bag (molecular weight cutoff: 1500 Da), dialyzed with deionized water (1 L) for 12 h, and the deionized water (1 L) was replaced every 4 hours; after the dialysis was completed, 5 g of the cross-linking agent 1,4-phenylenediboronic acid was added, stirred (450 rpm) for 6 h; the above solution was transferred to a dialysis bag (molecular weight cutoff of 1500Da) again and dialyzed with deionized water (1L) for 24 h, and the deionized water (1L) was replaced every 4 hours; after the dialysis was completed, the solution in the dialysis bag was homogenized by high-pressure microfluidization at a homogenization pressure of 80 MPa and twice; and after freeze-drying for 48 h, the EGCG preparation was obtained.

[0104] Embodiment 19

[0105] Polymer wall material PPEGMA 40 - b -PCL 20 - b -PPEGMA 40 The preparation steps are the same as in Example 1.

[0106] The prepared polymer wall material PPEGMA with a mass of 5 g 40 - b -PCL 20 - b -PPEGMA 40 2 g of EGCG preparation was added to 10 g of glycerol, heated to 60 °C, and stirred (speed: 500 rpm) for 30 min to obtain phase A solution; the phase A solution was slowly added dropwise (dropping speed: 8 mL / min) to 78 g of deionized water. After the addition was completed, stirring was continued (speed: 300 rpm) for 20 min; the mixed solution was transferred to a dialysis bag (molecular weight cutoff: 3000 Da), dialyzed with deionized water (1 L) for 12 h, and the deionized water (1 L) was replaced every 4 hours; after the dialysis was completed, 5 g of the cross-linking agent 1,4-phenylenediboronic acid was added, stirred (150 rpm) for 2 h; the above solution was transferred to a dialysis bag (molecular weight cutoff of 3000 Da) again and dialyzed with deionized water (1 L) for 24 h, and the deionized water (1 L) was replaced every 4 hours; after the dialysis was completed, the solution in the dialysis bag was homogenized by high-pressure microfluidization at a homogenization pressure of 50 MPa and 6 times; after freeze-drying for 48 h, the EGCG preparation was obtained.

[0107] Comparative Example 1 (without adding crosslinking agent)

[0108] Polymer wall material PPEGMA 40 - b -PCL 20 - b -PPEGMA 40 The preparation steps are the same as in Example 1.

[0109] The EGCG preparation was prepared by replacing 5 g of the cross-linking agent 1,4-phenylenediboric acid in Example 1 with 5 g of deionized water while keeping other conditions unchanged.

[0110] The difference between Comparative Example 1 and Example 1 is that no crosslinking modification treatment is performed in Comparative Example 1.

[0111] Comparative Example 2 (too little amount of cross-linking agent added, polymer: cross-linking agent = 1:0.3)

[0112] The EGCG preparation was prepared by replacing 5 g of the cross-linking agent 1,4-phenylenediboric acid in Example 1 with 1.5 g of 1,4-phenylenediboric acid, and replacing 68 g of deionized water with 71.5 g of deionized water, while keeping the other conditions unchanged.

[0113] Comparative Example 3 (too much cross-linking agent added, polymer: cross-linking agent = 1:2.5)

[0114] The EGCG preparation was prepared by replacing 5 g of the cross-linking agent 1,4-phenylenediboric acid in Example 1 with 12.5 g of 1,4-phenylenediboric acid, and replacing 68 g of deionized water with 60.5 g of deionized water, while keeping the other conditions unchanged.

[0115] Comparative Example 4 (MPEGMA replaces PEGMA, the MPEGMA molecule does not contain hydroxyl groups at the end, and no cross-linking reaction can occur even after adding a cross-linking agent)

[0116] (1) Polymer PMPEGMA 30 - b -PCL 20 - b -PMPEGMA 30 Preparation (taking the preparation of 1.0mmol as an example): Br-PCL 20 The preparation steps of Br-PCL are the same as those in Example 1. 20 -Br (2.67 g, 1.0 mmol), methoxy polyethylene glycol acrylate (M n =480, 28.80 g, 60.0 mmol), N,N,N',N',N''-pentamethyldiethylenetriamine (M n=173.30, 1.73 g, 10.0 mmol) was dissolved in tetrahydrofuran (100 mL) and the catalyst CuBr (M n =143.45, 0.29 g, 2.0 mmol), reacted at 60°C for 24 h, transferred the reaction mixture to a dialysis bag (molecular weight cutoff of 3000 Da), and dialyzed in deionized water (1 L) for 72 h, with the deionized water (1 L) replaced every 4 hours. After the dialysis, the liquid in the dialysis bag was freeze-dried for 24 h to obtain the polymer wall material PMPEGMA 30 - b -PCL 20 - b -PMPEGMA 30 (Number average molecular weight M n =31173).

[0117] (2) 5 g of the polymer wall material PPEGMA in Example 1 40 - b -PCL 20 - b -PPEGMA 40 and replaced with a polymer wall material PMPEGMA with a mass of 5g 30 - b -PCL 20 - b -PMPEGMA 30 , and the other conditions remained unchanged to prepare the EGCG preparation.

[0118] The difference between Comparative Example 4 and Example 1 is that the polymer in Comparative Example 4 cannot undergo a cross-linking reaction.

[0119] Comparative Example 5 (cross-linking first, then drug loading, EGCG cannot be encapsulated into the network polymer)

[0120] Polymer wall material PPEGMA 40 - b -PCL 20 - b -PPEGMA 40 The preparation steps are the same as in Example 1.

[0121] The prepared polymer wall material PPEGMA with a mass of 5 g 40 - b -PCL 20 - b -PPEGMA 40Add to 10g of 1,2-hexanediol, heat to 50℃, stir (speed: 300 rpm) for 60min to obtain phase A solution; slowly drop phase A solution (drop speed: 5mL / min) into 68g of deionized water, and continue to stir (speed: 300rpm) for 20min after the dropwise addition is completed; add 5g of cross-linking agent 1,4-phenylenediboronic acid to the mixed solution and stir (450rpm) for 4h; add 2g of EGCG to 10g of 1,2-hexanediol, heat to 50℃, and stir (speed: 300 rpm) for 60min; slowly drop EGCG alcohol solution (drop speed: 5mL / min) into the above mixed solution, and continue to stir (speed: 300 rpm) for 20 min; the above solution was transferred to a dialysis bag (molecular weight cutoff of 3000Da), and dialyzed with deionized water (2L) for 48h, and the deionized water (2L) was replaced every 4 hours; after the dialysis was completed, the solution in the dialysis bag was homogenized by high-pressure microfluidization, with a homogenization pressure of 80 MPa and a homogenization frequency of 4 times; and after freeze-drying for 48h, the EGCG preparation was obtained.

[0122] Comparative Example 6 (cross-linking and drug loading performed simultaneously)

[0123] Polymer wall material PPEGMA 40 - b -PCL 20 - b -PPEGMA 40 The preparation steps are the same as in Example 1.

[0124] The prepared polymer wall material PPEGMA with a mass of 5 g 40 - b -PCL 20 - b -PPEGMA 40 2 g of EGCG was added to 20 g of 1,2-hexanediol, heated to 50°C, and stirred (at a speed of 300 rpm) for 60 min to obtain a phase A solution; the phase A solution and 5 g of cross-linking agent 1,4-phenylenediboronic acid were slowly added dropwise (dropping speed: 5 mL / min) to 68 g of deionized water. After the addition was completed, stirring was continued (at 450 rpm) for 4 h; the above solution was transferred to a dialysis bag (molecular weight cutoff of 3000 Da), dialyzed with deionized water (2 L) for 48 h, and the deionized water (2 L) was replaced every 4 hours; after the dialysis was completed, the solution in the dialysis bag was subjected to high-pressure microfluidization homogenization with a homogenization pressure of 80 MPa and a homogenization number of 4 times; and after freeze-drying for 48 h, the EGCG preparation was obtained.

[0125] Comparative Example 7 (dialysis treatment was not used to remove the polyol solvent)

[0126] Polymer wall material PPEGMA 40 - b -PCL 20 - b -PPEGMA 40 The preparation steps are the same as in Example 1.

[0127] The prepared polymer wall material PPEGMA with a mass of 5 g 40 - b -PCL 20 - b -PPEGMA 40 2 g of EGCG was added to 20 g of 1,2-hexanediol, heated to 50 °C, and stirred (at a speed of 300 rpm) for 60 min to obtain a phase A solution; the phase A solution was slowly added dropwise (dropping speed: 5 mL / min) to 68 g of deionized water, and after the addition was completed, the stirring was continued (at a speed of 300 rpm) for 20 min; 5 g of cross-linking agent 1,4-phenylenediboronic acid was added and stirred (at 450 rpm) for 4 h; the above solution was transferred to a dialysis bag (molecular weight cutoff of 3000 Da) again, and dialyzed with deionized water (2 L) for 48 h, with the deionized water (2 L) being replaced every 4 hours; after the dialysis was completed, the solution in the dialysis bag was subjected to high-pressure microfluidization homogenization treatment, with a homogenization pressure of 80 MPa and a homogenization number of 4 times; and after freeze-drying for 48 h, the EGCG preparation was obtained.

[0128] Comparative Example 8 (polymer: EGCG = 1:2)

[0129] The EGCG preparation was prepared by replacing 2 g of EGCG in Example 1 with 6 g of EGCG, and replacing 68 g of deionized water with 64 g of deionized water, while keeping other conditions unchanged.

[0130] Comparative Example 9 (Traditional Technology Thin Film Liposome)

[0131] 5 g of soybean lecithin, 0.1 g of cholesterol and 2 g of EGCG were dissolved in 30 g of chloroform and stirred at 60 °C (speed of 150 rpm) for 3 h. The chloroform was removed from the mixed solution by rotary evaporation (40 °C, speed of 250 rpm). After the chloroform was completely removed, 92.9% deionized water was added, and the mixture was dispersed by ultrasound (600w) for 30 min. Finally, it was filtered through an organic microporous membrane with a pore size of 0.45 μm to obtain liposomes encapsulating EGCG.

[0132] Comparative Example 10 (Conventional Technology Ethanol Body)

[0133] 5g of soybean lecithin, 0.1g of cholesterol and 2g of EGCG were dissolved in 30g of 1,2-propylene glycol and stirred at 60°C (speed of 150 rpm) for 3h. The mixed solution was uniformly added to the phase B solution at a dropping speed of 5mL / min. After the addition was completed, stirring was continued (speed of 300 rpm) for 30min; then dispersed by high-speed shear (9000rpm) for 15min; and then subjected to high-pressure microfluidization homogenization treatment with a homogenization pressure of 80 MPa and a homogenization number of 4 times to obtain EGCG-encapsulated ethanolsomes.

[0134] Comparative Example 11 (EGCG aqueous solution)

[0135] 1.66 g of EGCG was dissolved in 99.17 g of deionized water, and stirred at 60° C. (rotation speed was 150 rpm) for 30 min to prepare an EGCG aqueous solution.

[0136] The content of EGCG in Comparative Example 11 is the same as the content of EGCG in the aqueous solution of Example 1 with a mass percentage of 10%.

[0137] Test Example 1 Storage stability test

[0138] The samples of Examples 1-19 and Comparative Examples 1-10 were prepared into 10% by mass aqueous solutions, and left to stand at room temperature (25°C) for one month, two months and three months, respectively. The particle size and encapsulation efficiency of the samples were then measured to test the storage stability of the samples.

[0139] Particle size characterization: Malvern Nano ZS90 particle size potential detector was used to characterize the particle size and particle size dispersion index (PDI) of the samples. The test angle was 90° and the test temperature was 25 o C, Each group of experiments was conducted three times in parallel, and the experimental results were taken as the arithmetic mean.

[0140] Active ingredient encapsulation rate: Take 200 m The sample aqueous solution with a mass percentage of 10% was ultrafiltrated and centrifuged (9000 rpm, 30 min) to obtain 5 m The filtrate was tested by high performance liquid chromatography (HPLC, Shimadzu, Japan) to determine the content of EGCG in the filtrate, i.e. the content of free EGCG in the EGCG preparation dispersed in the aqueous solution. A 10% sample aqueous solution was added with a mixed solution of methanol and deionized water, and ultrasonic demulsification was performed for 30 min at a ratio of sample: methanol: deionized water = 1:4:5 (v / v). m After filtering with organic filter membrane, take 5 mThe EGCG content of the sample solution was determined by high performance liquid chromatography (HPLC, Shimadzu, Japan) to obtain the total content of EGCG in the EGCG preparation. The encapsulation efficiency (EE) of the EGCG preparation was calculated according to formula (1).

[0141] Formula (1)

[0142] C1 represents the content of free EGCG in a sample aqueous solution with a mass percentage of 10%; C0 represents the total concentration of EGCG in the sample after ultrasonic demulsification of a mixed solution of methanol and deionized water.

[0143] Table 1 Particle size, encapsulation efficiency and storage stability test results of samples of Examples 1-19 and Comparative Examples 1-10

[0144]

[0145] Comparison of Examples 1-5 shows that different types of cross-linking agents (containing different numbers of benzene rings) ultimately affect the particle size, encapsulation efficiency and storage stability of the product. In Examples 1-5, the cross-linking agents are 1,4-phenylenediboric acid (containing 1 benzene ring), 4,4'-biphenylenediboric acid (containing 2 benzene rings), 2,6-dinaphthoboric acid (containing 2 benzene rings), anthracene-9,10-diboric acid (containing 3 benzene rings), B,B'-[1,1':4',1''-triphenyl]-4,4''-diylbis[boric acid] (containing 3 benzene rings). The results show that when the sample of Example 1 is stored for 3 months at room temperature (25°C), the change rate of its particle size and encapsulation efficiency is the smallest, which are +1.37% and -3.17%, respectively. Therefore, in the present invention, the cross-linking agent is selected from 1,4-phenylenediboric acid containing only 1 benzene ring in the molecular structure, and the storage stability of the EGCG preparation finally obtained is the best.

[0146] By comparing Example 1, Examples 6-8, and Comparative Examples 1-3, it can be seen that the addition amount of the crosslinking agent is different, which ultimately affects the particle size, encapsulation efficiency and storage stability of the product. In Example 1, Examples 6-8 and Comparative Examples 1-3, the feed ratios of polymer to crosslinking agent are 1:1.0, 1:0.5, 1:1.5, 1:2.0, 1:0, 1:0.3, and 1:2.5, respectively. As the addition amount of the crosslinking agent increases, the sample is stored at room temperature (25°C) for 3 months, and the change rates of its particle size and encapsulation efficiency both decrease and then increase. When the feed ratio of polymer to crosslinking agent is 1:1.0 (Example 1), the sample has the lowest change rates of particle size and encapsulation efficiency after being stored at room temperature (25°C) for 3 months, which are +1.37% and -3.17%, respectively. When the feed ratio of polymer to cross-linking agent is 1:0.3 (Comparative Example 2) and 1:2.5 (Comparative Example 3), the change rate of particle size and encapsulation efficiency is large. Therefore, the application selects the addition amount of cross-linking agent to be 1:0.5-2.0 (mass ratio, polymer: cross-linking agent), preferably 1:1.0.

[0147] By comparing Example 1 and Examples 9-10, it can be seen that the different polymerization degrees of PCL in the polymer structure ultimately affect the particle size, encapsulation efficiency and storage stability of the product. As the polymerization degree of PCL increases, the sample is stored at room temperature (25°C) for 3 months, and the change rates of its particle size and encapsulation efficiency both decrease and then increase. When the polymerization degree of PCL is 20 (Example 1), the change rates of the particle size and encapsulation efficiency of the sample after storage at room temperature (25°C) for 3 months are the lowest, which are +1.37% and -3.17%, respectively.

[0148] Comparing Example 1 and Examples 11-13, it can be seen that the polymerization degree of PPEGMA in the polymer structure is different, which ultimately affects the particle size, encapsulation efficiency and storage stability of the product. As the polymerization degree of PPPEGMA increases, the sample is stored at room temperature (25°C) for 3 months, and the change rate of its particle size and encapsulation efficiency decreases and then increases. When the polymerization degree of PPPEGMA is 40 (Example 1), the change rate of the particle size and encapsulation efficiency of the sample after storage for 3 months at room temperature (25°C) is the lowest, which is +1.37% and -3.17%, respectively.

[0149] By comparing Example 1, Examples 14-17, and Comparative Example 8, it can be seen that the feed ratio of the polymer to the active molecule EGCG is different, which ultimately affects the particle size, encapsulation rate, and storage stability of the product. In Example 1, Examples 14-17, and Comparative Example 8, the feed ratio of the polymer to the active molecule EGCG is 1:0.4, 1:0.2, 1:0.6, 1:0.8, 1:1.0, and 1:1.2, respectively. As the amount of active molecule EGCG added increases, the sample is stored at room temperature (25°C) for 3 months, and the change rate of its particle size and encapsulation rate both decreases and then increases. When the feed ratio of the polymer to the active molecule EGCG is 1:0.4 (Example 1), the sample is stored at room temperature (25°C) for 3 months. The change rate of the particle size and encapsulation rate is the lowest, which is +1.37% and -3.17%, respectively. When the feed ratio of the polymer to the active molecule EGCG is 1:1.2 (Comparative Example 8), the change rate of its particle size and encapsulation rate is large. Therefore, the present application selects a feed ratio of polymer to active molecule EGCG of 1:0.2-1.0, preferably 1:0.4.

[0150] Comparing Example 1 and Examples 18-19, the process parameters are different, which ultimately affect the particle size, encapsulation efficiency and storage stability of the product. When the process parameters in Example 1 are selected, the sample is stored at room temperature (25°C) for 3 months, and the change rates of its particle size and encapsulation efficiency are the lowest, that is, the storage stability of the sample in Example 1 is the best.

[0151] Comparison of Example 1 and Comparative Example 4 shows that, compared with MPEGMA, the polymer nano-wall material PPEGMA obtained by using PEGMA as the polymerization unit 40 - b -PCL 20 - b -PPEGMA 40 The storage stability of the sample finally obtained by encapsulating EGCG is optimal.

[0152] By comparing Example 1, Example 5 and Example 6, it can be seen that in the preparation process of EGCG preparation, the order of the drug loading process and the cross-linking reaction process is different, and the storage stability of the EGCG preparation finally obtained is different. In the preparation process of the sample of Example 1, the drug loading process is carried out first, and then the cross-linking process is carried out; in the preparation process of the sample of Example 5, the cross-linking process is carried out first, and then the drug loading process is carried out; in the preparation process of the sample of Example 6, the drug loading process and the cross-linking process are carried out simultaneously. The results show that in the preparation process of EGCG preparation, the polymer nano-wall material first encapsulates EGCG, and then the polymer is cross-linked with a cross-linking agent, which is beneficial to improve the storage stability of the sample.

[0153] By comparing Example 1 and Comparative Example 7, it can be seen that in the preparation process of the EGCG preparation, dialysis treatment was used to remove the polyol solvent in Example 1, while dialysis treatment was not used to remove the polyol solvent in Comparative Example 7; in contrast, the use of dialysis treatment to remove the polyol solvent in Example 1 is also beneficial to improving the storage stability of the sample.

[0154] Comparative Example 1, Comparative Example 9 and Comparative Example 10 show that, compared with the liposome and ethosome carrier forms in the prior art, the sample obtained by encapsulating EGCG with a polymer nano-wall material and then performing a cross-linking reaction with a cross-linking agent has the best storage stability.

[0155] Test Example 2 In vitro active substance release test

[0156] The in vitro active substance release test was conducted by dialysis method, and the specific method was as follows: phosphate buffer (pH 6.8) was used to prepare the sample into a solution with a mass percentage of 10%, 10 mL of the above sample solution with a mass percentage of 10% was taken and transferred to a dialysis bag (molecular weight cutoff of 3500Da), and phosphate buffer (pH 6.8), phosphate buffer containing 5mM H2O2 (pH 6.8) and phosphate buffer containing 10mM H2O2 (pH 6.8) were used as dialysis media, and the dialysis bag was immersed in different dialysis media (200 mL) for in vitro active substance release test, and the release conditions were maintained at a constant temperature of 37°C and a speed of 100r / min. At 0h, 1h, 2h, 4h and 8h, 4 mL of the dialysis medium in the release container was taken, marked and stored for testing, and 4 mL of fresh dialysis medium was added to the release container at the same time. The content of EGCG in the dialysis medium was determined by high performance liquid chromatography (HPLC, Shimadzu, Japan), and the cumulative release rate of the active substance in the sample was calculated according to formula (2): E r Each group of experiments was conducted three times in parallel, and the experimental results were taken as the arithmetic mean.

[0157] Formula (2)

[0158] V e represents the dialysis medium replenishment volume (4 mL); n Indicates the number of sampling times; C i (or C n ) indicates the i The second (or n times) the active substance concentration of the dialysis medium solution taken; V 0 represents the total volume of dialysis medium (210 mL); mIndicates the total mass of active substances in the sample. When the dialysis time is 1h, 2h, 4h and 8h, it corresponds to the first (i=1), second (i=2), third (i=3) and fourth (i=4) sampling respectively.

[0159] Table 2 In vitro active substance release rate of samples (%)

[0160]

[0161] As can be seen from Table 2, the type of cross-linking agent, the amount of cross-linking agent added, the order of the drug loading process and the cross-linking reaction process in the preparation of the EGCG preparation, and the type of dosage form will affect the release effect of the active ingredient in the sample.

[0162] As can be seen from Examples 1-8, compared with phosphate buffer, the in vitro release of EGCG in phosphate buffer containing H2O2 has a faster release rate and a higher release amount. And the higher the concentration of H2O2, the faster the release rate of EGCG of the sample and the higher the release amount, indicating that the EGCG preparation can quickly release the active substance EGCG phenol in a high active oxygen environment, and play an antioxidant effect. Compared with Comparative Examples 1-6, Comparative Example 9 and Comparative Example 10, the EGCG preparation prepared in Example 1 has the fastest release rate and the highest cumulative release rate of active substances in an environment with high concentration of H2O2.

[0163] Test Example 3 Transdermal absorption test

[0164] The transdermal absorption (permeation enhancement) experiment was used to evaluate the transdermal absorption (permeation enhancement) ability of EGCG preparations. The specific method is as follows:

[0165] A vertical diffusion cell was used for the in vitro transdermal experiment, with nude mouse skin as the model (abdominal skin, subcutaneous fat layer and blood vessels removed). The receiving solution was PBS solution. The skin piece was fixed between the supply cell and the receiving cell, with the skin layer facing up, and balanced for 20 minutes. Each sample was prepared into a sample solution with a mass percentage of 10%, and the solution was added to the supply cell. 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 at a ratio of 1:4:5 (V / V) for the receiving solution: methanol: water. 0.45 m After filtration with an 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 patch is as shown in formula (3):

[0166] Formula (3)

[0167] in, Qn for t Cumulative transmittance per unit area of ​​the sample ( m g / cm 2 ), A is the permeable area, C n for t The concentration of active substances is measured at time. C i for t The measured value of the active substance concentration sampled at the time point, V is the total volume of the receiving fluid, and V0 is the sampling volume at each time point.

[0168] Table 3 In vitro skin permeation test results of samples

[0169]

[0170] As shown in Table 3, by comparing Examples 1-5, it can be seen that the types of cross-linking agents (containing different numbers of benzene rings) are different, and the transdermal performance of the EGCG preparations finally prepared is different. The results show that when the cross-linking agent is 1,4-phenylenediboronic acid (containing 1 benzene ring), the cumulative permeation per unit area of ​​the EGCG preparation (Example 1) prepared at 1h, 4h, 8h and 24h is 3.12 m g / cm 2 , 15.54 m g / cm 2 , 35.43 m g / cm 2 , 95.16 m g / cm 2 . They are all higher than the samples of Examples 2-5, indicating that the transdermal performance of Example 1 is the best.

[0171] By comparing Example 1, Examples 6-8, and Comparative Examples 1-3, it can be seen that the amount of cross-linking agent added is different, and the transdermal performance of the EGCG preparation finally prepared is different. The results show that when the feed ratio of polymer to cross-linking agent is selected as 1:1, the cumulative permeation per unit area of ​​the EGCG preparation (Example 1) finally prepared is the highest, indicating that the transdermal performance of Example 1 is the best. In addition, compared with the cross-linking reaction without adding a cross-linking agent (Comparative Example 1), the present application uses 1,4-phenylenediboronic acid cross-linking agent to cross-link the polymer wall material, and the EGCG preparation (Example 1) finally prepared has a higher cumulative permeation per unit area, indicating that its transdermal performance is better.

[0172] Comparison of Example 1 and Comparative Example 4 shows that, compared with MPEGMA, the polymer nano-wall material PPEGMA obtained by using PEGMA as the polymerization unit 40 - b -PCL20 - b -PPEGMA 40 The EGCG preparation (Example 1) finally prepared by encapsulating EGCG has a higher cumulative permeation per unit area, indicating that its transdermal performance is better.

[0173] Compared with Comparative Example 7 (dialysis treatment was not used), the present application used dialysis treatment to remove the polyol solvent, and the EGCG preparation (Example 1) finally prepared had a higher cumulative permeation per unit area, indicating that its transdermal performance was better.

[0174] Compared with the carrier forms of liposomes (Comparative Example 9) and ethosomes (Comparative Example 10) in the prior art, and the EGCG aqueous solution without encapsulation treatment (Comparative Example 11), the present application uses polymer nano-wall materials to encapsulate EGCG, and then uses a cross-linking agent to carry out a cross-linking reaction. The EGCG preparation (Example 1) finally prepared has the highest cumulative permeation per unit area, indicating that its transdermal performance is optimal.

[0175] Test Example 4 Antioxidant-Cellular Reactive Oxygen Species ROS Inhibition Test

[0176] The samples of the embodiment and the comparative example were respectively prepared into sample solutions with a mass percentage of 10% using PBS buffer (pH 6.8).

[0177] Human skin keratinocytes (HaCaT cells) were digested and prepared into a cell suspension, which was then inoculated into a 96-well plate at 1.0 × 10 5 -2.0×10 5 The blank group (PBS buffer was added, but no light was applied), the control group (PBS buffer was added, but light was applied) and the experimental group (the samples of the embodiment and the comparative example were added, respectively) were cultured using DMEM medium (Shanghai MacLean Biochemical Technology Co., Ltd.). After 24 hours of culture, the control group and the experimental group were exposed to UVA light (the light dose was 6-10 J / cm 2 ), after the illumination, the culture medium was replaced, and 100 μL of different test samples (experimental group) or PBS solution (blank group and control group) were added, and the culture was continued for 2h and 4h. The cell culture medium was removed, washed with PBS 3 times, and 100 μL of 10 μmol / L DCFH-DA (reactive oxygen fluorescence probe, Shanghai Biyuntian Biotechnology Co., Ltd.) was added to each well and placed in a CO2 incubator for 20 min. After the incubation, PBS was washed 3 times, and serum-free DMEM culture medium was added again. The ROS content was detected 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 is the comparison of the OD value of the experimental group (or the OD value of the control group) with the OD value of the blank group.

[0178] Table 4 Relative levels of ROS in HaCaT cells after light damage in sample treatment

[0179]

[0180] As can be seen from Table 4, the relative ROS levels of the EGCG preparations prepared in Examples 1-8 for HaCaT cells damaged by light are lower than those of the control group, indicating that they have good antioxidant properties and can effectively remove reactive oxygen species in cells. In addition, the relative ROS levels of the EGCG preparations prepared in Examples 1-8 for HaCaT cells damaged by light are lower than those in Comparative Examples 1-7 and Comparative Examples 9-11, indicating that Examples 1-8 have better antioxidant properties, among which the EGCG preparation in Example 1 has the lowest relative ROS level for HaCaT cells damaged by light, and after co-incubation with the HaCaT cells damaged by light for 4h and 8h, the relative ROS levels are 102.8% and 101.5%, respectively, which are close to the normal level of cells not damaged by light, indicating that the EGCG preparation in Example 1 has the best antioxidant properties, and it can quickly exert its antioxidant effect in a short time (2h), quickly and effectively remove reactive oxygen species in HaCaT cells damaged by light, and effectively reduce the damage of reactive oxygen species to cells.

[0181] Test Example 5 DNA repair damage - anti-photoaging

[0182] DNA double-strand breaks are considered the most serious DNA damage. H2AX, the full name of which is H2A histone family member X, can also be referred to as H2AX, which is one of the variants of chromosome histone H2A. When the double-strand DNA breaks in cells, H2AX will be phosphorylated to form phosphorylated H2AX, i.e. γ-H2AX. The γ-H2AX content level can clearly reflect the degree of DNA damage and repair, and is widely used in DNA damage and cell apoptosis research, becoming an important DNA damage marker.

[0183] In this test case, the DNA damage repair experiment was performed using a DNA damage detection kit (γ-H2AX immunofluorescence method, mouse monoclonal antibody, green), which can be purchased directly from Shanghai Biyuntian Biotechnology Co., Ltd. Human skin keratinocytes (HaCaT cells) were digested and prepared into a cell suspension, which was then inoculated into a 96-well plate, with 1.0×10 5 -2.0×10 5 The cells were cultured in DMEM medium for 24 hours, and then the blank control group and the experimental group were exposed to UVA light (light dose of 6-10J / cm 2). After the illumination, the culture medium was replaced, and 100 μL of different test samples (experimental group) or PBS solution (blank group and control group) were added respectively, and the culture was continued for 2h and 4h. The cell culture medium was removed and washed with PBS 3 times. The cells were collected by centrifugation and washed once with PBS. After the PBS was completely aspirated, the cells were appropriately flicked. The fixative solution prepared in the kit was added, the cells were gently suspended, and fixed for 10 minutes. Centrifugation was performed, the fixative solution was removed, and the washing solution prepared in the kit was added to wash once. A small amount of washing solution was used to resuspend the cells, and the cells were added dropwise to the coverslip or slide to make a smear. After fully drying, the washing solution was used to wash twice, 1 mL of immunostaining blocking solution was added, and the cells were blocked at room temperature for 15 minutes. The immunostaining blocking solution was removed, and 1 mL of γ-H2AX mouse monoclonal antibody was added, and the cells were incubated at room temperature for 1 hour. The cells were washed 3 times with the washing solution, and 1 mL of anti-mouse 488 was added, and the cells were incubated at room temperature for 1 hour. The cells were washed 2 times with the washing solution, 1 mL of nuclear staining solution (DAPI) was added, and the cells were stained at room temperature for 5 minutes, the nuclear staining solution was removed, and the cells were washed 3 times with the washing solution. Finally, the fluorescence intensity value of the sample was measured for analysis.

[0184] The results of DNA damage repair (γ-H2AX method) of Example 1, Comparative Example 1 and Comparative Example 11 (EGCG aqueous solution) are as follows Figure 1 As shown. The results show. After the cells were irradiated with radiation (ultraviolet rays), the fluorescence intensity was significantly increased compared with normal cells, indicating that radiation (ultraviolet rays) caused DNA damage. After treatment with Example 1, Comparative Example 1, and Comparative Example 11 (EGCG aqueous solution), the fluorescence intensity decreased, indicating that the experimental group (Example 1, Comparative Example 1, Comparative Example 11) had a repair effect on cell DNA damage. Compared with Comparative Example 1 and Comparative Example 11, after treatment with Example 1, the fluorescence intensity in the cells was significantly reduced in a short time (2h and 4h), indicating that the EGCG preparation in Example 1 has a high efficiency in repairing DNA-damaged cells, and the repair effect is better than that of the EGCG preparation (Comparative Example 1) and the EGCG aqueous solution (Comparative Example 11) that have not been cross-linked and modified.

[0185] Test Example 6 ATP generation capacity test

[0186] Mitochondria are important regulators of cell energy and metabolism, and play a vital role in maintaining cell growth and survival. The core function of mitochondria is to synthesize ATP through oxidative phosphorylation. As the most important energy molecule, ATP plays an important role in various physiological and pathological processes of cells. Changes in ATP levels directly affect cell function. Usually, a decrease in ATP levels indicates that mitochondrial function is impaired or decreased. During cell apoptosis, a decrease in ATP levels usually occurs simultaneously with a decrease in mitochondrial membrane potential.

[0187] The ATP production capacity test in this test example uses an ATP detection kit, which can be purchased directly from Shanghai Biyuntian Biotechnology Co., Ltd. The specific experimental method is as follows: (1) Preparation of ATP standard solution: Melt the reagent to be used in an ice bath, and dilute the ATP standard solution with ATP detection lysis buffer to a certain concentration gradient (0.1nM, 0.3nM, 1nM, 3nM, 10nM, 30nM, 100nM). (2) Sample measurement preparation: Digest human skin keratinocytes (HaCaT cells) and make a cell suspension, then inoculate it into a 6-well plate, 5.0×10 per well. 5 -10.0×10 5 The cells were cultured in DMEM medium for 24 hours, and then the blank control group and the experimental group were exposed to UVA light (light dose of 6-10 J / cm 2 After the illumination, the culture medium was replaced and different test samples (experimental group) or PBS solution (blank group and control group) were added. The culture was continued for 2h and 4h. The cell culture medium was removed and the cells were washed 3 times with PBS. The cells were collected by centrifugation and washed once with PBS. After the PBS was completely aspirated, the cells were appropriately dispersed. 200 μL of PBS was added according to the number of cells in each well. m L of lysis buffer, lyse the cells, centrifuge at 4°C for 5 min (12,000 rpm), and take the supernatant for testing. (3) Determination of ATP concentration: Add 100 μL of lysis buffer to the test tube. m L of ATP detection working solution, place at room temperature for 3-5 minutes to consume all the background ATP, thereby reducing the background. m L of the sample to be tested or ATP standard solution, mix with a micropipette, and measure the RLU value with a chemiluminescence instrument. Draw an ATP standard curve with the ATP content of the ATP standard solution as the horizontal axis and the RLU value as the vertical axis. The ATP content of the sample to be tested can be calculated based on the ATP standard curve.

[0188] The results of ATP generation capacity of Example 1, Comparative Example 1 and Comparative Example 11 (EGCG aqueous solution) are as follows: Figure 2 As shown. The results show. After the cells were irradiated with radiation (ultraviolet rays), the ATP content in the cells decreased significantly, indicating that the function of the mitochondria in the cells was damaged or decreased. After treatment with Example 1, Comparative Example 1, and Comparative Example 11 (EGCG aqueous solution), the ATP content increased, indicating that the experimental group (Example 1, Comparative Example 1, Comparative Example 11) has the effect of promoting ATP generation. Compared with Comparative Example 1 and Comparative Example 11, after treatment with Example 1, the ATP content in the cells increased significantly in a short time (2h and 4h), indicating that the EGCG preparation in Example 1 can effectively promote the generation of ATP in cells, and the effect is better than the EGCG preparation (Comparative Example 1) and the EGCG aqueous solution (Comparative Example 11) that have not been cross-linked and modified.

[0189] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing an EGCG preparation with ultra-high stability and high anti-aging efficacy, characterized in that: The EGCG is first encapsulated by a polymer wall material, and then an esterification reaction is carried out with a cross-linking agent containing two boric acid groups to obtain an EGCG preparation; the specific steps include: S1: adding the polymer wall material and EGCG to the polyol, heating and stirring to obtain a phase A solution; S2: adding the phase A solution dropwise into water, and after the addition is complete, continuing to stir to obtain a mixed solution, removing the polyol from the mixed solution, adding a crosslinking agent, and stirring sufficiently to obtain a crosslinked polymer mixed solution; S3: performing nano-dispersion treatment on the cross-linked polymer mixed solution, and then freeze-drying it to obtain an EGCG preparation; The mass ratio of the polymer wall material to EGCG in step S1 is 1:0.4±0.1; the mass ratio of the polymer wall material to the polyol is 1:2-10; The cross-linking agent described in step S2 is 1,4-phenylenediboronic acid; The mass ratio of the polymer wall material to the cross-linking agent is 1:1±0.5; the mass ratio of the polymer wall material to EGCG is 1:0.2-1.0; The polymer wall material has amphiphilicity and is composed of polymerized monomers. ε -caprolactone is obtained by ring-opening polymerization to obtain polycaprolactone, which is then brominated to obtain brominated polycaprolactone, which is then obtained by atom transfer radical polymerization of the polymer monomer poly(ethylene glycol) methacrylate; the molecular weight of the polymer wall material is M n =32000-63000; the degree of polymerization of the polycaprolactone is 40±20.

2. The preparation method according to claim 1, characterized in that: The polyol is at least one of 1,3-propylene glycol, glycerol, 1,4-butanediol, pentanediol, 1,2-hexanediol, 1,6-hexanediol, octyldodecanol, ethylene glycol, dipropylene glycol, and ethoxydiglycol.

3. The preparation method according to claim 2, characterized in that: The heating temperature in step S1 is 30-60°C, the stirring speed is 100-500 rpm, and the stirring time is 30-90 min; In step S2, the dropping speed is 1-10 mL / min; the stirring speed is 100-400 rpm; the stirring time is 10-30 min; the cross-linked polymer mixed solution is dialyzed with water; the polyol in the mixed solution is dialyzed with water, and the dialysis conditions are: the molecular weight cutoff of the dialysis bag used is 500-5000 Da; the dialysis time is 12-48h; the water is replaced every 2-4h; The nano-dispersion treatment described in step S3 includes high-speed shear dispersion treatment and / or microfluidization homogenization treatment. The high-speed shear dispersion treatment is performed by shear homogenization using a high-speed shear homogenizer with a rotation speed of 6000-12000 rpm and a shear dispersion time of 10-20 min; the microfluidization homogenization treatment is performed by homogenization using a microfluidizer with a homogenization pressure of 50-120 MPa and a homogenization cycle number of 1-8 times.

4. The preparation method according to claim 1, 2 or 3, characterized in that: The method for preparing the polymer wall material comprises the following steps: (1) ε -caprolactone is added to a polyol terminated with hydroxyl groups at both ends, and a tin catalyst is added to the reaction system under the protection of an inert gas, and the reaction is carried out at 80-160°C for 8-18 hours to prepare polycaprolactone; (2) dissolving the polycaprolactone in an organic solvent, adding 2-bromoisobutyryl bromide under the protection of an inert gas, reacting at -5-5°C for 2-4 hours, and then continuing to react at 25±15°C for 12-48 hours to obtain brominated polycaprolactone; (3) Dissolving the polymerization monomers poly(ethylene glycol) methacrylate, brominated polycaprolactone, and N,N,N',N',N''-pentamethyldiethylenetriamine in an organic solvent, adding a copper salt catalyst to the reaction system under the protection of an inert gas, and reacting at 40-80° C. for 12-48 hours to prepare the polymer wall material.

5. The preparation method according to claim 4, characterized in that: The polyol with hydroxyl groups at both ends in step (1) is at least one of 1,4-butanediol, 1,5-pentanediol, and 1,6-hexanediol; the tin catalyst is at least one of stannous isooctanoate and stannous octoate; ε The molar ratio of -caprolactone to the catalyst is 1:0.005-0.02; the polyol initiator having hydroxyl groups at both ends and ε -The molar ratio of caprolactone is 1:10-40; The molar ratio of polycaprolactone to 2-bromoisobutyryl bromide in step (2) is 1:4.0-12.0; The organic solvent in steps (2) and (3) includes at least one of tetrahydrofuran, chloroform, N,N-dimethylformamide and dimethyl sulfoxide; In step (3), the molar ratio of the brominated polycaprolactone to the polymerizable monomers poly(ethylene glycol) methacrylate, N,N,N',N',N''-pentamethyldiethylenetriamine and the copper salt catalyst is 1:50-150:5-15:0.5-5; the copper salt catalyst is at least one of CuBr, CuCl and CuI.

6. The EGCG preparation with ultrahigh stability and high anti-aging efficacy obtained by the method according to any one of claims 1 to 5.

7. Use of the EGCG preparation with ultrahigh stability and high anti-aging efficacy as claimed in claim 6 in cosmetics, or in the preparation of external skin medicines.

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