Nano-composition based on EGCG with powerful antioxidant and anti-photoaging effects, preparation process and applications thereof

Abstract

Nano-composition based on EGCG with potent antioxidant and anti-photoaging effects, preparation method and applications thereof. The present invention proposes a nano-composition based on EGCG with potent antioxidant and anti-photoaging effects, a preparation method and applications thereof. The EGCG-based nano-composition is prepared by encapsulating the functional active substance EGCG through self-assembly with functionalized phospholipids in two steps. First, a precursor of the nano-composition is prepared to encapsulate the EGCG, and then the nano-composition precursor is self-assembled with the phospholipids for protection, thus obtaining the EGCG-based nano-composition. The EGCG-based nano-composition exhibits excellent stability, high encapsulation efficiency, antioxidant effects, prolonged release, and good skin permeability.It also offers effective DNA damage repair and inhibits protein carbonylation, which enhances antioxidant, anti-photoaging and brightening effects in dermal formulations, surpassing aqueous EGCG solution.

Description

Title of the invention: Nano-composition based on EGCG with powerful antioxidant and anti-photoaging effects, method of preparation and applications thereof. Technical field

[0001] The present invention belongs to the technical field of nanocarrier materials, and relates more particularly to a nano-composition based on EGCG with powerful antioxidant and anti-photoaging effects, as well as a preparation process and its applications. TECHNICAL CONTEXT

[0002] As the largest organ of the human body, the skin is directly exposed to the external environment, and when the skin is exposed to harmful stimuli such as air pollution, oxidation, and long-term ultraviolet radiation, signs of skin aging often occur. Skin aging is mainly manifested by dryness and sagging of the skin, wrinkles, pigmentation, and other manifestations. Thus, "anti-aging" always attracts people's attention. Among these, yellowing of the skin is mainly caused by the accumulation of carbonylated proteins in the skin; therefore, inhibiting protein carbonylation can effectively improve the problem of skin yellowing.

[0003] Epigallocatechin gallate (EGCG) is a catechin compound, which is the most abundant component of catechins, representing a percentage of 50 to 60%. EGCG has a special stereochemical structure and exhibits extremely potent antioxidant activity as well as a significantly greater free radical scavenging action than vitamin C and vitamin E, which can protect cells and DNA from damage, thus delaying skin aging. However, when EGCG is used as an anti-aging additive in cosmetic products, it has drawbacks such as low stability, discoloration, poor transdermal absorption, and low bioavailability.For example, cosmetic products containing EGCG often exhibit discoloration, due to the fact that EGCG's molecular structure is rich in phenolic hydroxyl groups. These groups are easily oxidized under photothermal conditions, leading to the formation of quinones, which ultimately causes the products to redden and weakens their anti-aging effect. Secondly, given its high water solubility, EGCG has low transdermal absorption and bioavailability, and cannot penetrate the skin effectively. the keratic layer of the skin, with a penetrability of only 0.44% (Drug Delivery, 2017, 24: 61-74).

[0004] To improve the stability and percutaneous penetration of EGCG, EGCG is generally encapsulated in the prior art. Chunhua Liu (South China Agricultural University, 2020) proposed an EGCG-based nanoparticle prepared by encapsulating EGCG using sodium caseinate and acacia gum as nanocarrier wall materials and investigated its application in antioxidant activity in gastric juice and intestinal fluid. However, the encapsulation yield of the EGCG-based nanoparticle using this technology was low, at only 62.8%. Liu Hong (Zhejiang Industrial and Commercial University, 2015) proposed an EGCG-based nanoethosome prepared by an ultrasonic method to enhance the anti-UV effect. Sun Xiang (Jiangnan University, 2019) proposed an EGCG-based liposome prepared by ethanol injection.The two existing methods described above both use absolute ethanol as a solvent, which can cause skin irritation when applied to the skin. Furthermore, most existing EGCG encapsulation technologies often present problems such as low encapsulation efficiency and high irritation. In addition, most existing EGCG encapsulation technologies focus solely on the physical and chemical properties (such as particle size, encapsulation yield, etc.) of the encapsulated products and basic research in the anticancer field; no studies on the application of EGCG-based encapsulated products in improving photoaging and skin yellowing have been reported. DISCLOSURE OF THE INVENTION

[0005] To remedy the above drawbacks in the existing art, a first object of the present invention is to provide a method for preparing a nano-composition based on EGCG with powerful antioxidant and anti-photoaging effects.

[0006] Another object of the present invention is to provide a nano-composition based on EGCG having powerful antioxidant, anti-photoaging and protein carbonylation inhibitor effects, obtained by the above preparation process.

[0007] The present invention also provides applications of the EGCG-based nano-composition with powerful antioxidant, anti-photoaging and protein carbonylation inhibitor effects, obtained by the above preparation process.

[0008] The objects of the present invention are expected by the following technical solution:

[0009] The EGCG-based nanocomposition according to the present invention is prepared by encapsulating the functional active substance EGCG by self-assembly of functionalized phospholipids in two steps, including a first step of preparation of a precursor of the nano-composition and a second step of preparation of a double phospholipid nano-composition.

[0010] A process for preparing a nano-composition based on EGCG with powerful antioxidant and anti-photoaging effects comprises the following steps:

[0011] (1) Preparation of a precursor of the nano-composition:

[0012] Add a phospholipid and EGCG to an organic solvent and stir uniformly to obtain an organic phase; add a polyol to water and stir uniformly to obtain an aqueous phase; add the organic phase dropwise to the aqueous phase at a constant rate, and once the addition is complete, stir again to obtain a mixed solution; remove the organic solvent from the mixed solution; and perform a drying treatment to obtain a precursor of the nano-composition;

[0013] Expressed as a mass percentage, the phospholipid content in the mixed solution is from 1% to 10%;

[0014] (2) Preparation of a nano-composition with a double phospholipid:

[0015] Add a phospholipid, a rigid membrane stabilizer and a flexible membrane stabilizer to the polyol, and stir uniformly to obtain an alcoholic phase; add the precursor of the nano-composition to water, stir and disperse until a homogeneous dispersion is obtained to obtain an aqueous phase; add the aqueous phase dropwise to the alcoholic phase at a constant rate, and once the addition is complete, stir again to obtain a mixed solution; and perform a nano-treatment on the mixed solution to obtain an EGCG-based nano-composition, the EGCG representing a mass percentage of 0.6% to 6.0% of the EGCG-based nano-composition;

[0016] Expressed as a mass percentage, the phospholipid content in the mixed solution is 0.1% to 1.0%, the rigid membrane stabilizer content is 0.1% to 0.3% and the flexible membrane stabilizer content is 0.5% to 2.0%;

[0017] The molecular structure of said rigid membrane stabilizer comprises a polycyclic structure unit or a benzene ring-type structure unit; the molecular structure of said flexible membrane stabilizer is a linear chain structure or a linear chain compound.

[0018] Preferably, the rigid membrane stabilizer comprises one or more of cholesterol, phytosterol, sodium cholate, vitamin D, resorcinol, phenylalanine and phenylpropanol.

[0019] Preferably, the flexible membrane stabilizer comprises one or more of the following: caprylic / capric triglyceride, caprylic / capric / linoleic acid triglyceride, caprylic / capric / lauric triglyceride, oleic acid, linoleic acid, octyldodecanol and octyldodecyl oleate.

[0020] Preferably, EGCG represents a mass percentage of 1% to 5% of the EGCG-based nanocomposition.

[0021] Preferably, expressed as a mass percentage, in step (1), the phospholipid content in the mixed solution is 3% to 7%, the EGCG content is 10% to 20%, the polyol content is 1% to 10%, the organic solvent content is 10% to 50% and the water content is 13% to 78%.

[0022] Preferably, expressed as a mass percentage, in step (2), the phospholipid content in the mixed solution is 0.1% to 0.5%, the rigid membrane stabilizer content is 0.1% to 0.2%, the flexible membrane stabilizer content is 1% to 2%, the polyol content is 5% to 50%, the precursor content of the nano-composition is 1.0% to 10% and the water content is 37.3% to 88.4%.

[0023] Preferably, the phospholipid in step (1) is at least one of soy lecithin, hydrogenated soy lecithin, enzymatically hydrolyzed lecithin, dipalmitoylphosphatidylcholine, palmitoylphosphatidylglycerol and egg yolk lecithin.

[0024] Preferably, the phospholipid in step (2) is at least one of soy lecithin, hydrogenated soy lecithin, dipalmitoylphosphatidylcholine, palmitoylphosphatidylglycerol and egg yolk lecithin.

[0025] Preferably, the organic solvent in step (1) is one or more of absolute ethanol, methanol, dichloromethane, trichloromethane, acetone, tetrahydrofuran, n-hexane, petroleum ether and ethyl acetate.

[0026] Preferably, the polyol in step (1) is a sugar alcohol, which is one or more of maltitol, D-sorbitol, xylitol, erythritol, mannitol and lactitol.

[0027] Preferably, the polyol in step (2) is at least one of glycerol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,2-hexanediol, ethoxydiethylene glycol, dipropylene glycol and polyethylene glycol (Mn = 400).

[0028] Preferably, the conditions of the first two stirrings in step (1): the stirring speed is 100 to 800 rpm; the stirring temperature is 30 to 80°C; the stirring time is 20 to 60 min; and the drop-by-drop addition rate is 1 to 10 ml / min; the conditions of the third stirring: the stirring speed is 100 to 800 rpm; the stirring temperature is 30 to 80°C; the stirring time is 10 to 30 min; the organic solvent is removed from the mixed solution by rotary evaporation at a rotary evaporation speed of 100 to 350 rpm; the rotary evaporation temperature is 30 to 80°C; and the rotary evaporation time is 1 to 3 h. The drying method can be freeze-drying, vacuum drying and air drying, preferably freeze-drying; the drying time is 24 to 72 hours.

[0029] Preferably, the conditions of the first two stirrings in step (2): the stirring speed is 100 to 800 rpm; the stirring temperature is 10 to 50°C; the The stirring time is 20 to 60 minutes; and the drop-by-drop addition rate is 1 to 10 ml / min; the conditions for the third stirring: the stirring speed is 100 to 800 rpm; the stirring temperature is 10 to 50°C; and the stirring time is 10 to 30 minutes.

[0030] Preferably, the nano-treatment in step (2) comprises high-speed shear dispersion and / or microfluid homogenization; the rotation speed of the high-speed shear dispersion is 6000 to 12000 rpm and the shear dispersion time is 10 to 30 min; a homogenization pressure of the microfluid homogenization is 50 to 120 MPa and a number of homogenization cycles is 2 to 6.

[0031] The mode of agitation in steps (1) and (2) is mechanical agitation or magnetic agitation.

[0032] Compared with existing techniques, the present invention has the following advantages and beneficial effects:

[0033] (1) The present invention employs an encapsulation of the active substance Functional EGCG is produced by self-assembly with functionalized phospholipids in two steps. This involves first preparing a precursor of the nanocomposition to encapsulate the EGCG, and then self-assembling the nanocomposition precursor with phospholipids for protection, thus obtaining the EGCG-based nanocomposition. The EGCG-based nanocomposition according to the present invention exhibits good stability, good encapsulation efficiency, good antioxidant effect, good sustained-release capacity, and good permeability.

[0034] (2) In the present invention, two membrane stabilizers with properties Different are used to adjust the composition of phospholipid membranes, therefore the resulting EGCG-based nanocomposition exhibits improved stability, encapsulation yield, antioxidant effect, prolonged release capacity and percutaneous permeability.

[0035] (3) The EGCG-based nanocomposition according to the present invention also has a good DNA damage repair effect and a good protein carbonylation inhibition effect, and can play an antioxidant, anti-photoaging and anti-yellowing role in topical dermal formulations with better effects than those of aqueous EGCG-based solutions. DESCRIPTION OF THE FIGURES

[0036] [Fig.1] is a view illustrating the results of DNA damage repair by the y-H2AX method of the present invention.

[0037] [Fig.2] is a view illustrating the results of the carbonylation inhibition test proteins of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention will be described in more detail below with reference to the embodiments, but the implementation of the present invention is not limited to those embodiments. Unless otherwise clearly stated, the conditions in the embodiments are the conventional conditions or the conditions specified by the manufacturer. Unless otherwise clearly stated, the reagents or instruments are commercial products purchased on the market. Unless otherwise clearly stated, the reagents used in the embodiments can be readily purchased on the market.

[0039] In embodiments of the present invention, the EGCG (98% purity), maltitol (98% purity), D-sorbitol (98% purity), xylitol (98% purity) used can be purchased directly from the Aladdin Biotechnology Co., Ltd. of Shanghai. Example 1

[0040] (1) Preparation of the precursor of the nano-composition: add 5.0% Add 15% by mass of soy lecithin and 15% by mass of EGCG to 30% by mass of absolute ethanol, and stir (at 300 rpm) at 60°C for 30 minutes until the above raw materials are completely dissolved to obtain an organic phase. Add 5% by mass of maltitol to 45% by mass of demineralized water, and stir (at 300 rpm) at 60°C for 30 minutes until the above raw materials are completely dissolved to obtain an aqueous phase. Maintaining the temperature at 60°C, add the above organic phase dropwise to the aqueous phase at a constant rate of 3 mL / min, and once the dropwise addition is complete, stir again at 60°C (at 300 rpm) for 15 minutes to obtain a mixed solution.Remove absolute ethanol from the mixed solution by rotary evaporation at a rotary evaporation speed of 150 rpm, a rotary evaporation temperature of 40 °C, and a rotary evaporation time of 2 h. Lyophilize for 48 hours to obtain a precursor of the nano-composition. In the nano-composition precursor, expressed as mass percentages, phospholipid: EGCG: maltitol = 20%: 60%: 20%.

[0041] (2) Preparation of the nano-composition: add 0.3% by mass percentage of hydrogenated lecithin, 0.15% by mass percentage of cholesterol and 1.5% by mass percentage of caprylic / capric acid triglyceride to a mixed solution composed of 25% by mass percentage of glycerol and 10% in Add a 5% mass percentage of 1,2-hexanediol to 58.05% mass percentage of demineralized water and stir at 30°C for 30 minutes until the above raw materials are completely dissolved to obtain an alcoholic phase. Stir at 30°C for 30 minutes until the nano-composition precursor is homogeneously dispersed to obtain an aqueous phase. Maintaining the temperature at 30°C, add the aqueous phase dropwise to the alcoholic phase at a constant rate of 3 mL / min. Once the dropwise addition is complete, stir again at 30°C (300 rpm) for 15 minutes to obtain a mixed solution.Disperse the mixed solution by high-speed shearing (10000 rpm) for 20 min; then perform high-pressure microfluidic homogenization, with a homogenization pressure of 80 MPa and a homogenization cycle number of 4, thus obtaining the EGCG-based nano-composition.

[0042] In example 1 of the present invention, the functional active substance EGCG represents a percentage of 3% in the EGCG-based nano-composition.

[0043] Example 2 (examples 2 to 5, the quantities of phosphatide added in the nanocomposition were different)

[0044] (1) Preparation of the precursor of the nano-composition: prepare the precursor of The nano-composition is created by substituting 1% by mass percentage of soy lecithin for 5% by mass percentage of soy lecithin from Example 1, substituting 49% by mass percentage of demineralized water for 45% by mass percentage of demineralized water, and keeping the other components unchanged. In the precursor of the nano-composition in this example, expressed as mass percentages, phospholipid: EGCG: maltitol = 4.8% : 71.4% : 23.8%.

[0045] (2) Preparation of the EGCG-based nano-composition: prepare the nano EGCG-based composition by substituting 4.2% by mass percentage of precursor of the nano-composition prepared above with 5% by mass percentage of precursor of the nano-composition of example 1, and keeping the other components unchanged.

[0046] In example 2 of the present invention, in the EGCG-based nano-composition, the functional active substance EGCG represents the same percentage as in example 1, i.e. 3%. Example 3

[0047] (1) Preparation of the precursor of the nano-composition: prepare the precursor of the nano-composition by substituting 3% by mass percentage of soy lecithin for 5% by mass percentage of soy lecithin from example 1, substituting 47% by mass percentage of demineralized water for 45% by mass percentage of water demineralized, and now the other components unchanged. In the precursor of the nano-composition of the present example, expressing in mass percentage, phospholipid: EGCG: maltitol = 13%: 65.2%: 21.8%.

[0048] (2) Preparation of the EGCG-based nano-composition: prepare the nano EGCG-based composition by substituting 4.6% by mass percentage of precursor of the nano-composition prepared above with 5% by mass percentage of precursor of the nano-composition of example 1, and keeping the other components unchanged.

[0049] In example 3 of the present invention, in the EGCG-based nano-composition, the functional active substance EGCG represents the same percentage as in example 1, i.e. 3%. Example 4

[0050] (1) Preparation of the precursor of the nano-composition: prepare the precursor of The nano-composition is created by substituting 7% by mass percentage of soy lecithin for 5% by mass percentage of soy lecithin from Example 1, substituting 43% by mass percentage of demineralized water for 45% by mass percentage of demineralized water, and keeping the other components unchanged. In the precursor of the nano-composition in this example, expressed as mass percentages, phospholipid: EGCG: maltitol = 25.9%: 55.6%: 18.5%.

[0051] (2) Preparation of the EGCG-based nano-composition: prepare the nano EGCG-based composition by substituting 5.4% by mass percentage of precursor of the nano-composition prepared above with 5% by mass percentage of precursor of the nano-composition of example 1, and keeping the other components unchanged.

[0052] In example 4 of the present invention, in the EGCG-based nano-composition, the functional active substance EGCG represents the same percentage as in example 1, i.e. 3%. Example 5

[0053] (1) Preparation of the precursor of the nano-composition: prepare the precursor of the Nanocomposition by substituting 10% by mass percentage of soy lecithin for 5% by mass percentage of soy lecithin from Example 1, substituting 40% by mass percentage of demineralized water for 45% by mass percentage of demineralized water, and keeping the other components unchanged. In the precursor of the nanocomposition of the present example, expressed as mass percentages, phospholipid: EGCG: maltitol = 33.3% : 50% : 16.7%.

[0054] (2) Preparation of the EGCG-based nano-composition: prepare the nano composition based on EGCG by substituting 6% by mass percentage of precursor of the nano-composition prepared above at 5% by mass percentage of precursor of the nano-composition of example 1, and now the other components unchanged.

[0055] In example 3 of the present invention, in the EGCG-based nano-composition, the functional active substance EGCG represents the same percentage as in example 1, i.e. 3%.

[0056] Example 6 (examples 6 to 9, the quantities of hydrogenated lecithin added were different)

[0057] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0058] (2) Substitute 0.1% by mass percentage of hydrogenated lecithin for 0.3% in For the mass percentage of hydrogenated lecithin in Example 1, substitute 60.25% by mass percentage of demineralized water with 60.05% by mass percentage of demineralized water, and keep the other components unchanged for a total mass percentage of 100%. Prepare the EGCG-based nanocomposition with the following treatments unchanged.

[0059] In example 6 of the present invention, the functional active substance EGCG represents a percentage of 3% in the EGCG-based nano-composition. Example 7

[0060] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0061] (2) Substitute 0.5% by mass percentage of hydrogenated lecithin for 0.3% in For the mass percentage of hydrogenated lecithin in Example 1, substitute 59.85% by mass percentage of demineralized water with 60.05% by mass percentage of demineralized water, and keep the other components unchanged for a total mass percentage of 100%. Prepare the EGCG-based nanocomposition with the following treatments unchanged.

[0062] In example 7 of the present invention, the functional active substance EGCG represents a percentage of 3% in the EGCG-based nano-composition. Example 8

[0063] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0064] (2) Substitute 0.7% by mass percentage of hydrogenated lecithin for 0.3% in For the mass percentage of hydrogenated lecithin in Example 1, substitute 59.65% by mass percentage of demineralized water with 60.05% by mass percentage of demineralized water, and keep the other components unchanged. total mass of 100%. Prepare the EGCG-based nanocomposition with the following treatments unchanged.

[0065] In example 8 of the present invention, the functional active substance EGCG represents a percentage of 3% in the EGCG-based nano-composition. Example 9

[0066] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0067] (2) Substitute 1.0% by mass percentage of hydrogenated lecithin for 0.3% in For the mass percentage of hydrogenated lecithin in Example 1, substitute 59.35% by mass percentage of demineralized water with 60.05% by mass percentage of demineralized water, and keep the other components unchanged for a total mass percentage of 100%. Prepare the EGCG-based nanocomposition with the following treatments unchanged.

[0068] In example 9 of the present invention, the functional active substance EGCG represents a percentage of 3% in the EGCG-based nano-composition.

[0069] Example 10 (examples 10 to 12, the types of rigid stabilizer were different)

[0070] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0071] (2) Prepare the EGCG-based nanocomposition by substituting 0.15% in mass percentage of sodium cholate at 0.15% in mass percentage of cholesterol from example 1, and now the other components unchanged.

[0072] In example 10 of the present invention, the functional active substance EGCG represents a percentage of 3% in the EGCG-based nano-composition. Example 11

[0073] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0074] (2) Prepare the EGCG-based nanocomposition by substituting 0.15% in mass percentage of resorcinol at 0.15% in mass percentage of cholesterol from example 1, and now the other components unchanged.

[0075] In example 11 of the present invention, the functional active substance EGCG represents a percentage of 3% in the EGCG-based nano-composition. Example 12

[0076] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0077] (2) Prepare the EGCG-based nanocomposition by substituting 0.15% in mass percentage of phenylalanine to 0.15% in mass percentage of cholesterol from example 1, and now the other components unchanged.

[0078] In example 12 of the present invention, the functional active substance EGCG represents a percentage of 3% in the EGCG-based nano-composition.

[0079] Example 13 (examples 13 to 15, the quantities of rigid stabilizer added were different)

[0080] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0081] (2) Substitute 0.1% by mass percentage of cholesterol for 0.15% by percentage For the mass percentage of cholesterol in Example 1, substitute 60.10% by mass percentage of demineralized water with 60.05% by mass percentage of demineralized water, and keep the other components unchanged for a total mass percentage of 100%. Prepare the EGCG-based nanocomposition with the following treatments unchanged.

[0082] In Example 13 of the present invention, the functional active substance EGCG represents 3% of the EGCG-based nanocomposition. Example 14

[0083] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0084] (2) Substitute 0.2% by mass percentage of cholesterol for 0.15% by percentage For the mass percentage of cholesterol in Example 1, substitute 60.00% by mass percentage of demineralized water with 60.05% by mass percentage of demineralized water, and keep the other components unchanged for a total mass percentage of 100%. Prepare the EGCG-based nanocomposition with the following treatments unchanged.

[0085] In Example 14 of the present invention, the functional active substance EGCG represents 3% of the EGCG-based nanocomposition. Example 15

[0086] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0087] (2) Substitute 0.3% by mass percentage of cholesterol for 0.15% by percentage For the mass percentage of cholesterol in Example 1, substitute 59.90% by mass percentage of demineralized water with 60.05% by mass percentage of demineralized water, and keep the other components unchanged for a total mass percentage of 100%. Prepare the EGCG-based nanocomposition with the following treatments unchanged.

[0088] In example 15 of the present invention, the functional active substance EGCG represents a percentage of 3% in the EGCG-based nano-composition.

[0089] Example 16 (examples 16 to 18, the types of flexible stabilizer were different)

[0090] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0091] (2) Prepare the EGCG-based nanocomposition by substituting 1.5% in mass percentage of caprylic / capric / linoleic acid triglyceride at 1.5% in mass percentage of caprylic / capric acid triglyceride from example 1, and now the other components unchanged.

[0092] In example 16 of the present invention, the functional active substance EGCG represents a percentage of 3% in the EGCG-based nano-composition. Example 17

[0093] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0094] (2) Prepare the EGCG-based nanocomposition by substituting 1.5% in mass percentage of linoleic acid at 1.5% in mass percentage of caprylic / capric acid triglyceride from example 1, and now the other components unchanged.

[0095] In example 17 of the present invention, the functional active substance EGCG represents a percentage of 3% in the EGCG-based nano-composition. Example 18

[0096] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0097] (2) Prepare the EGCG-based nanocomposition by substituting 1.5% in octyldodecanol mass percentage at 1.5% in caprylic / capric acid triglyceride mass percentage of example 1, and now the other components unchanged.

[0098] In example 18 of the present invention, the functional active substance EGCG represents a percentage of 3% in the EGCG-based nano-composition.

[0099] Example 19 (examples 19 to 21, the quantities of flexible stabilizer were different)

[0100] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0101] (2) Substitute 0.5% by mass percentage of caprylic acid triglyceride / 1.5% caprylic / capric triglyceride (by mass percentage) of Example 1, substituted 61.05% by mass percentage of demineralized water with 60.05% by mass percentage of demineralized water, and kept the other components unchanged for a total mass percentage of 100%. Prepare the EGCG-based nanocomposition with the following treatments unchanged.

[0102] In example 19 of the present invention, the functional active substance EGCG represents a percentage of 3% in the EGCG-based nano-composition. Example 20

[0103] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0104] (2) Substitute 1.0% by mass percentage of caprylic acid triglyceride / 1.5% caprylic / capric triglyceride (by mass percentage) of Example 1, substituted 60.55% by mass percentage of demineralized water with 60.05% by mass percentage of demineralized water, and keeping the other components unchanged for a total mass percentage of 100%. Prepare the EGCG-based nanocomposition with the following treatments unchanged.

[0105] In example 20 of the present invention, the functional active substance EGCG represents a percentage of 3% in the EGCG-based nano-composition. Example 21

[0106] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0107] (2) Substitute 2.0% by mass percentage of caprylic acid triglyceride / 1.5% caprylic / capric triglyceride (by mass percentage) of Example 1, substituted 59.55% by mass percentage of demineralized water with 60.05% by mass percentage of demineralized water, and kept the other components unchanged for a total mass percentage of 100%. Prepare the EGCG-based nanocomposition with the following treatments unchanged.

[0108] In example 21 of the present invention, the functional active substance EGCG represents a percentage of 3% in the EGCG-based nano-composition.

[0109] Example 22 (examples 22 to 23, the quantities of precursor of the nano-composition added were different)

[0110] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0111] (2) Substitute 1% by mass percentage of precursor of the nano-composition To a 5% by mass percentage precursor of the nano-composition of Example 1, substitute 64.05% by mass percentage of demineralized water with 60.05% by mass percentage of demineralized water, and keep the other components unchanged for a total mass percentage of 100%. Prepare the EGCG-based nano-composition with the following treatments unchanged.

[0112] In example 22 of the present invention, the functional active substance EGCG represents a percentage of 0.6% in the EGCG-based nano-composition. Example 23

[0113] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0114] (2) Substitute 10% by mass percentage of precursor of the nano-composition To a 5% by mass percentage precursor of the nano-composition of Example 1, substitute 55.05% by mass percentage of demineralized water with 60.05% by mass percentage of demineralized water, and keep the other components unchanged for a total mass percentage of 100%. Prepare the EGCG-based nano-composition with the following treatments unchanged.

[0115] In example 23 of the present invention, the functional active substance EGCG represents a percentage of 6% in the EGCG-based nano-composition.

[0116] Example 24 (examples 24 to 25, the types and quantities of other materials added were different)

[0117] The difference between the present example and example 1 is that:

[0118] (1) The precursor of the nano-composition comprises the components in percentage The following components are used by mass: dipalmitoylphosphatidylcholine 5%, EGCG 10%, methanol 10%, D-sorbitol 5%, and demineralized water 70%. The precursor of the nanocomposition is obtained by keeping the other components unchanged. In the precursor of the nanocomposition in this example, expressed as mass percentages, dipalmitoylphosphatidylcholine : EGCG : D-sorbitol = 25% : 50% : 25%.

[0119] (2) The EGCG-based nanocomposition comprises the components in The following components are present by mass percentage: Palmitoylphosphatidylglycerol 0.3%, cholesterol 0.15%, caprylic / capric triglyceride 1.5%, 1,3-propanediol 5%, ethoxydiethylene glycol 5%, nano-composition precursor 5%, and demineralized water 83.05%. The EGCG-based nano-composition is obtained while keeping the other components unchanged.

[0120] In example 24 of the present invention, the functional active substance EGCG represents a percentage of 2.5% in the EGCG-based nano-composition. Example 25

[0121] The difference between the present example and example 1 is that:

[0122] (1) The precursor of the nano-composition comprises the components in percentage The following components are used by mass: egg yolk lecithin 5%, EGCG 20%, trichloromethane 50%, xylitol 5%, and demineralized water 20%. The precursor of the nano-composition is obtained while keeping the other components unchanged. In the nano-composition of this example, expressed as mass percentages, egg yolk lecithin : EGCG : xylitol = 25% : 66.7% : 25%.

[0123] (2) The EGCG-based nanocomposition comprises the components in The following mass percentages are present: dipalmitoylphosphatidylcholine 0.3%, cholesterol 0.15%, caprylic / capric triglyceride 1.5%, 1,2-butanediol 35%, polyethylene glycol (Mn = 400) 15%, precursor of the nano-composition 5%, and demineralized water. 43.05%. The EGCG-based nano-composition is obtained while keeping the other components unchanged.

[0124] In example 25 of the present invention, the functional active substance EGCG represents a percentage of 3.3% in the EGCG-based nano-composition.

[0125] Comparative Example 1 (Traditional Liposome - Soy Lecithin - Monolayer)

[0126] Prepare a traditional liposome by the thin membrane method using soy lecithin as the wall material. By weight of loaded materials, dissolve 1.5 g of soy lecithin, 0.15 g of cholesterol and 3 g of EGCG in 30 g of absolute ethanol, stir (at a speed of 300 rpm) for 30 min until complete dissolution of the above raw materials to obtain an alcoholic phase. Remove absolute ethanol from the alcohol phase by rotary evaporation at a rotary evaporation speed of 150 rpm, a rotary evaporation temperature of 40 °C and a rotary evaporation time of 2 h, thus forming a light yellow phospholipid membrane layer on the wall of the rotary evaporation tank.Add 95.35 g of demineralized water to the rotary evaporation tank, agitate (at a speed of 300 rpm) for 30 min to hydrate the phospholipid membrane until the phospholipid membrane on the tank wall is completely detached, thus obtaining a traditional EGCG liposome.

[0127] In comparative example 1 of the present invention, in the EGCG-based nano-composition, the functional active substance EGCG represents the same percentage as in example 1, i.e. 3%.

[0128] Comparative example 2 (Traditional liposome - hydrogenated lecithin - monolayer)

[0129] Prepare a traditional liposome by the thin-membrane method using hydrogenated lecithin as the wall material. By weight of loaded materials, dissolve 1.5 g of hydrogenated lecithin, 0.15 g of cholesterol, and 3 g of EGCG in 30 g of absolute ethanol, and stir (at a speed of 300 rpm) for 30 min until the above raw materials are completely dissolved to obtain an alcoholic phase. Remove the absolute ethanol from the alcoholic phase by rotary evaporation at a rotary evaporation speed of 150 rpm, a rotary evaporation temperature of 40 °C, and a rotary evaporation time of 2 h, thus forming a light yellow phospholipid membrane layer on the wall of the rotary evaporation tank.Add 95.35 g of demineralized water to the rotary evaporation tank, agitate (at a speed of 300 rpm) for 30 min to hydrate the phospholipid membrane until the phospholipid membrane on the tank wall is completely detached, thus obtaining a traditional EGCG liposome.

[0130] In comparative example 2 of the present invention, in the EGCG-based nano-composition, the functional active substance EGCG represents the same percentage as in example 1, i.e. 3%. Comparative example 3 (only rigid ligand added)

[0131] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0132] (2) Prepare the EGCG-based nanocomposition by substituting 1.5% in mass percentage of demineralized water at 1.5% in mass percentage of caprylic / capric acid triglyceride of example 1, and now the other components unchanged and the following treatments unchanged.

[0133] Compared to example 1, the difference is that only the rigid membrane stabilizer, i.e. cholesterol, is added in comparative example 3, without a flexible membrane stabilizer. Comparative example 4 (only flexible ligand added)

[0134] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0135] (2) Prepare the EGCG-based nanocomposition by substituting 0.15% in mass percentage of demineralized water at 0.15% mass percentage of cholesterol from example 1, and now the other components unchanged and the following treatments unchanged.

[0136] Compared to example 1, the difference is that only the flexible membrane stabilizer, namely caprylic / capric acid triglyceride, is added in comparative example 4, without a rigid membrane stabilizer.

[0137] Comparative example 5 (without added membrane stabilizer)

[0138] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0139] (2) Prepare the EGCG-based nanocomposition by substituting 1.65% in mass percentage of demineralized water at 0.15% mass percentage of cholesterol and 1.5% mass percentage of caprylic / capric acid triglyceride from example 1, and now the other components unchanged and the following treatments unchanged.

[0140] Compared to example 1, the difference is that no membrane stabilizer is added in comparative example 5.

[0141] Comparative example 6 (1 EGCG was added during the self-assembly process with phospholipids in the second step)

[0142] (1) Prepare the EGCG-based nanocomposition by substituting 15% in mass percentage of demineralized water at 15% in mass percentage of EGCG from example 1, and now the other components unchanged.

[0143] (2) add 0.3% by mass percentage of hydrogenated lecithin, 0.15% by mass percentage Add 1.5% by mass of cholesterol, 1.5% by mass of caprylic / capric triglyceride, and 3% EGCG to a mixed solution composed of 25% by mass of glycerol and 10% by mass of 1,2-hexanediol, and stir (at a speed of 300 rpm) at 30°C for 30 min until the above raw materials are completely dissolved to obtain an alcoholic phase. Maintaining the temperature at 30°C, add 60.05% by mass of demineralized water dropwise to the alcoholic phase at a constant rate of 3 ml / min, and once the dropwise addition is complete, stir again at 30°C (at a speed of 300 rpm) for 15 min to obtain a mixed solution.Disperse the mixed solution by high-speed shearing (10000 rpm) for 20 min; then perform high-pressure microfluidic homogenization, with a homogenization pressure of 80 MPa and a homogenization cycle number of 4, thus obtaining the EGCG-based nano-composition.

[0144] In comparative example 6 of the present invention, the functional active substance EGCG represents a percentage of 3% in the EGCG-based nano-composition.

[0145] Comparative example 7 (The encapsulation technique was not used, and EGCG was additionally added after the preparation of the EGCG-based nanocomposition)

[0146] (1) Prepare the EGCG-based nanocomposition by substituting 15% in mass percentage of demineralized water at 15% in mass percentage of EGCG from example 1, and now the other components unchanged.

[0147] (2) The steps for preparing the nano-composition are identical to those of Example 1.

[0148] (3) Add 3% by mass percentage of EGCG to 97% by mass percentage of nano-composition, and shake (at a speed of 300 rpm) at 30°C for 30 min to obtain a composition based on EGCG.

[0149] Comparative example 8 (1 ' EGCG n ' was encapsulated with phospholipids in a single step, without precursor of the nano-composition)

[0150] Add 0.3% by mass percentage of hydrogenated lecithin, 0.15% by mass percentage of cholesterol, 1.5% by mass percentage of caprylic / capric acid triglyceride and 3% of EGCG to a mixed solution composed of 25% by mass percentage of glycerol and 10% by mass percentage of 1,2-hexanediol, and shake (at a speed of 300 rpm) for 30 min until The above raw materials must be completely dissolved to obtain an alcoholic phase. Add the above alcoholic phase dropwise to 60.05% by mass of demineralized water at a constant rate of 3 ml / min, and once the dropwise addition is complete, continue stirring (at a speed of 300 rpm) for 15 min to obtain a mixed solution. Disperse the mixed solution by high-speed shearing (10,000 rpm) for 20 min; then perform high-pressure microfluidic homogenization with a homogenization pressure of 80 MPa and 4 homogenization cycles. The EGCG-based nanocomposition is obtained.

[0151] Compared to example 1, the difference is that the EGCG-based nanocomposition was prepared by encapsulation with phospholipids in a single step in comparative example 8. In comparative example 8 of the present invention, the functional active substance EGCG represents a percentage of 3% in the EGCG-based nanocomposition.

[0152] Comparative example 9 (1' EGCG was dissolved in the aqueous phase, then mixed with the alcoholic phase)

[0153] (1) Preparation of the precursor of the nano-composition: add 5.0% Add 30% by mass of soy lecithin to 45% by mass of absolute ethanol and stir (at 300 rpm) at 60°C for 30 minutes until the above raw materials are completely dissolved to obtain an organic phase. Add 5% by mass of maltitol and 15% by mass of EGCG to 45% by mass of demineralized water, and stir (at 300 rpm) at 60°C for 30 minutes until the above raw materials are completely dissolved to obtain an aqueous phase. Maintaining the temperature at 60°C, add the organic phase dropwise to the aqueous phase at a constant rate of 3 mL / min, and once the dropwise addition is complete, stir again at 60°C (at 300 rpm) for 15 minutes to obtain a mixed solution.Remove absolute ethanol from the mixed solution by rotary evaporation at a rotary evaporation speed of 150 rpm, a rotary evaporation temperature of 40 °C, and a rotary evaporation time of 2 h. Lyophilize for 48 hours to obtain a precursor of the nano-composition. In the nano-composition precursor, expressed as mass percentages, phospholipid: EGCG: maltitol = 20%: 60%: 20%.

[0154] (2) The steps for preparing the EGCG-based nanocomposition were identical to those in example 1.

[0155] Compared to example 1, the difference is that during the preparation of the precursor of the nano-composition in comparative example 9, the EGCG was first dissolved in demineralized water and then mixed with the alcoholic phase.

[0156] Comparative example 10 (Surface modification with chitosan)

[0157] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0158] (2) Prepare the nano-composition based on EGCG modified with chitosan by substituting 0.3% by mass percentage of chitosan for 0.3% by mass percentage of hydrogenated lecithin, and keeping the other components unchanged and the following treatments unchanged.

[0159] Compared to example 1, the difference is that the precursor of the nanocomposition was modified with chitosan in comparative example 10.

[0160] Comparative example 11 (Modification of the surface with gum arabic)

[0161] (1) The steps for preparing the precursor of the nano-composition are identical to those in example 1.

[0162] (2) Prepare the nano-composition based on the modified EGCG with the gum arabic by substituting 0.3% by mass percentage of gum arabic for 0.3% by mass percentage of hydrogenated lecithin, and keeping the other components unchanged and the following treatments unchanged.

[0163] Compared to example 1, the difference is that the precursor of the nanocomposition was modified with gum arabic in comparative example 11.

[0164] Comparative example 12 (Nano-emulsion based on 1' EGCG)

[0165] Mix 3% by mass percentage of EGCG and 15% by mass percentage of caprylic / capric acid triglyceride and heat to 85°C until the EGCG is completely dissolved, then add 10% by mass percentage of butanediol to obtain the components of the oil phase. Add the above oil phase dropwise to 72% by mass percentage of demineralized water at a constant rate of 3 ml / min, and once the dropwise addition is complete, stir again (at a speed of 300 rpm) for 15 min to obtain a mixed solution. Disperse the mixed solution by high-speed shearing (10,000 rpm) for 20 min; then perform high-pressure microfluid homogenization, with a homogenization pressure of 80 MPa and a number of homogenization cycles of 4, thus obtaining a nano-emulsion based on EGCG.

[0166] Comparative example 13 (Aqueous solution of 1' EGCG at 3%)

[0167] Add 3% by mass percentage of EGCG to 97% by mass percentage of demineralized water, and stir (at a speed of 300 rpm) for 15 min until complete dissolution of the EGCG to obtain an aqueous solution of EGCG.

[0168] Example of test 1 (particle size test)

[0169] The particle size of the samples from Examples 1 to 25 and Comparative Examples 1 to 12 was characterized using a Malvern Nano-ZS90 dynamic light scattering particle size analyzer, with a test angle of 90° and At a test temperature of 25°C, three parallel tests were performed for each group, and arithmetic mean values ​​were calculated on the test results.

[0170] Example of test 2 (test of the encapsulation yield of 1' EGCG)

[0171] EGCG encapsulation yield tests were carried out on samples from Examples 1 to 25 and Comparative Examples 1 to 12. 200 qL of sample of the EGCG-based nanocomposition was taken, ultrafiltration centrifuged (9000 rpm, 30 min), and 5 qL of filtrate was taken and the EGCG content determined using a high-performance liquid chromatograph (HPLC, Shimadzu, Japan), i.e. the content of EGCG not encapsulated in the EGCG-based nanocomposition.Take another sample of the EGCG-based nano-composition described above, add a methanol-water mixture (methanol:water = 1:4:5 v / v), demulsify by ultrasound for 30 min, filter through a 0.45 µm organic filter membrane, take 5 µL of the sample solution, and determine the EGCG content using high-performance liquid chromatography (HPLC, Shimadzu, Japan). This determines the EGCG content in the EGCG-based nano-composition. Calculate the EGCG encapsulation yield (EE) of the EGCG-based nano-composition according to formula (1). The analysis column used in the HPLC system was a non-polar C18 column, the mobile phase was acetonitrile, the flow rate was 1.0 ml / min and the column temperature was 30 °C. Three parallel tests were performed for each group and arithmetic mean values ​​were calculated on the test results. ...C. ... = (1 - -^jx (Formula 1 )

[0172] Ci represents the concentration of unencapsulated EGCG in the sample; Co represents the concentration of EGCG in the sample after ultrasonic demulsification with the mixed solution of methanol and demineralized water.

[0173] Example test 3 (storage stability test)

[0174] After leaving the samples of examples 1 to 25 and comparative examples 1 to 12 at room temperature for 1 month, 2 months and 3 months, the particle size and encapsulation yield of the samples were determined respectively by the methods described in test examples 1 and 2, and the storage stability of the samples was tested.

[0175] Table 1 Characterization of particle size and encapsulation yield of samples, as well as storage stability at room temperature for 0 to 3 months

[0176] [Tables] Samples Storage time (months) Particle size (nm) Rate of change in particle size (%) Encapsulation yield (%) Rate of change in encapsulation yield (%) Example 1 0 83.4 — 89.2 — 1 82.1 1.46 88.1 1.18 2 81.1 2.66 86.4 3.14 3 80.2 3.77 85.4 4.31 Example 2 0 105.2 — 77.7 — 1 98.7 6.16 74.1 4.61 2 93.8 10.87 69.8 10.23 3 91.6 12.93 67.3 13.40 Example 3 0 97.7 — 80.8 — 1 92.3 5.49 78.1 3.33 2 88.4 9.50 74.5 7.77 3 85.4 12.53 72.0 10.82 Example 4 0 126.2 — 73.1 — 1 116.2 7.98 68.5 6.28 2 108.8 13.81 65.4 10.57 3 104.1 17.55 62.6 14.40 Example 5 0 169.4 — 67.4 — 1 151.9 10.30 61.9 8.14 2 143.8 15.12 59.6 11.63 3 136.0 19.70 Example 6: 0 88.4 — 68.5 — 1 84.2 4.80 63.1 8.01 2 80.5 8.96 60.0 12.42 3 77.5 12.31 54.8 20.10 Example 7: 0 105.3 — 78.5 — 1 98.2 6.69 74.5 5.14 2 92.8 11.85 68.9 12.27 Example 8: 0 134.2 — 74.3 — 1 124.3 7.39 68.5 7.69 2 117.6 12.40 64.2 13.57 3 112.7 16.03 60.6 18.40 Example 9: 0 145.9 — 70.0 — 1 131.6 9.81 64.5 7.84 2 123.7 15.24 60.0 14.24 3 117.0 19.79 56.3 19.52 Example 10: 0 102.9 — 78.7 — 1 98.9 3.89 74.7 5.12 2 97.2 5.52 70.9 9.96 3 95.2 7.47 69.2 12.09 Example 11 0 98.0 — 67.8 — 1 95.0 3.10 62.3 8.10 2 93.2 4.90 58.6 13.55 3 89.1 9.06 56.9 16.03 Example 12 0 106.9 — 74.1 — 1 100.6 5.91 69.0 6.95 2 97.5 8.76 65.3 11.93 3 96.0 10.18 63.7 14.05 Example 13 0 88.5 — 68.9 — 1 83.1 6.06 64.9 5.85 2 79.8 9.76 62.6 9.11 3 74.9 15.37 59.9 13.01 Example 14 0 107.0 — 74.3 — 1 100.7 5.83 70.3 5.28 2 96.9 9.42 67.3 9.35 3 93.0 13.09 66.2 10.86 Example 15 0 125.0 — 70.3 — 1 116.1 7.11 66.2 5.89 2 112.7 9.80 63.3 10.03 3 104.6 16.26 60.7 13.69 Example 16 0 98.8 — 76.6 — 1 92.4 6.48 72.2 5.70 2 89.5 9.39 71.2 7.02 3 86.9 12.06 67.4 11.99 Example 17 0 103.1 — 74.1 — 1 96.1 6.83 69.9 5.74 2 93.0 9.78 67.3 9.21 3 84.6 18.00 63.7 14.05 Example Example 18 0 105.1 — 72.5 — 1 95.7 8.98 66.8 7.88 2 90.2 14.23 64.4 11.13 3 85.8 18.42 61.0 15.77 Example 19 0 120.4 — 68.8 — 1 114.3 5.05 63.3 7.88 2 110.8 7.97 61.1 11.13 3 106.2 11.83 57.9 15.77 Example 20 0 105.1 — 76.4 — 1 98.7 6.09 72.0 5.72 2 95.4 9.21 69.2 9.38 3 88.9 15.45 64.9 15.10 Example 21 0 112.6 — 67.5 — 1 104.6 7.04 60.8 9.95 2 98.6 12.37 56.8 15.92 3 93.6 16.84 54.0 20.07 Example 22 0 97.0 — 67.6 — 1 91.0 6.19 61.8 8.61 2 88.7 8.50 60.0 11.26 3 84.6 12.71 55.1 18.54 Example 23 0 133.4 — 71.3 — 1 124.2 6.95 66.3 7.06 2 116.5 12.71 64.3 9.89 3 112.2 15.95 61.1 14.29 Example 24 0 134.0 — 65.4 — 1 118.0 11.94 57.6 11.99 2 108.6 18.93 53.4 18.32 3 103.7 22.63 50.2 23.29 Example 25 0 150.7 — 70.2 — 1 134.8 10.56 64.0 8.93 2 122.7 18.58 60.4 14.04 3 111.4 26.06 57.3 18.34 Comparative example 1 0 164.5 — 41.5 — 1 197.2 19.88 31.5 24.10 2 217.2 32.04 28.4 31.57 3 230.5 40.12 13.8 66.75 Comparative example 2 0 212.7 — 38.7 — 1 263.4 23.84 27.8 28.17 2 287.2 35.03 22.4 42.12 3 304.5 43.16 12.4 67.96 Comparative example 3 0 212.8 — 48.6 — 1,270.2 26.97 38.6 20.58 2,297.1 39.61 34.9 28.19 3,329.8 54.98 27.7 43.00 Comparative example 4 0 143.7 — 40.3 — 1,178.2 24.01 29.8 26.05 2,198.3 38.00 26.6 34.00 3,218.4 51.98 21.5 46.65 Comparative example 5 0 155.6 — 34.5 — 1 202.2 29.95 24.6 28.70 2 220.9 41.97 20.6 40.29 3 245.8 57.97 16.7 51.59 Comparative example 6 0 148.6 — 55.4 — 1 175.3 17.97 43.2 22.02 2 190.4 28.13 36.1 34.84 3 197.8 33.11 32.1 42.06 Comparative example 7 0 137.2 — 22.8 — 1 161.9 18.00 13.5 40.79 2 172.8 25.95 8.2 64.04 3 186.6 36.01 4.8 78.95 Comparative example 8 0 155.3 — 53.7 — 1 189.6 22.09 41.5 22.72 2 203.4 30.97 34.4 35.94 3 214.3 37.99 30.1 43.95 Comparative example 9 0 188.6 — 50.3 — 1 246.8 30.86 38.1 24.25 2 254.3 34.84 32.2 35.98 3 272.6 44.54 26.5 47.32 Comparative example 10 0 212.8 — 43.6 — 1,294.6 38.44 31.4 27.98 2,312.3 46.76 26.2 39.91 3,353.1 65.93 18.8 56.88 Comparative example 11 0 201.6 — 48.2 — 1,274.1 35.96 35.7 25.93 2,286.3 42.01 27.8 42.32 3,328.5 62.95 21.2 56.02 Comparative example 12 0 415.8 — 40.2 — 1,540.1 29.89 27.6 31.34 2 602.6 44.93 22.1 45.02 3 664.3 59.76 14.8 63.18

[0177] Table 1 shows the results of the particle size characterization tests and encapsulation yield of the samples from Examples 1 to 25 and Comparative Examples 1 to 12, as well as the variation in particle size and encapsulation yield of the samples from Examples 1 to 25 and Comparative Examples 1 to 12 after being left at room temperature for 1 month, 2 months and 3 months.

[0178] By comparing examples 1 to 5, it can be seen that in the precursors of the nanocomposition, the quantities of soy lecithin added were different, and that the particle size, encapsulation yield and storage stability of the EGCG-based nanocompositions finally prepared were different.In examples 1 to 5, the mass percentage of soy lecithin was 5%, 1%, 3%, 7% and 10% respectively. With increasing amounts of soy lecithin added to the nano-composition precursor, the particle size of the finally prepared EGCG-based nano-composition decreased first and then increased, and the encapsulation yield increased first and then decreased. When the mass percentage of soy lecithin in the nano-composition precursor was 5% (example 1), the particle size of the finally prepared EGCG-based nano-composition was the smallest, at 83.4 nm, and the encapsulation yield was the highest, at 89.2%.Furthermore, the storage stability of the EGCG-based nanocomposition from Example 1 was better than that of Examples 2 through 5, and when the sample was stored at room temperature for three months, the rate of change in particle size and the rate of change in encapsulation yield of the EGCG-based nanocomposition were the lowest, at 3.77% and 4.31%, respectively. Therefore, the sample from Example 1 exhibited the best EGCG encapsulation effect.

[0179] Comparing Examples 1 and 6 to 9, it can be seen that because the amounts of hydrogenated lecithin added differed, the particle sizes, encapsulation yield, and storage stability of the ultimately prepared EGCG-based nanocompositions were different. In Examples 1 and 6 to 9, the mass percentage of hydrogenated lecithin was 0.3%, 0.1%, 0.5%, 0.7%, and 1.0%, respectively. With increasing amounts of hydrogenated lecithin added, the particle size of the EGCG-based nanocomposition gradually increased, while the encapsulation yield initially increased and then decreased. When the mass percentage of hydrogenated lecithin in the EGCG-based nanocomposition was 0.3% (example 1), the encapsulation effect of EGCG was best.

[0180] Comparing Examples 1 and 10 to 12, it can be seen that because the types of rigid membrane stabilizer differed, the particle sizes, encapsulation yield, and storage stability of the ultimately prepared EGCG-based nanocompositions varied. In Examples 1 and 10 to 12, the rigid membrane stabilizer was cholesterol, sodium cholate, resorcinol, and phenylalanine, respectively. The results show that when cholesterol was chosen as the rigid membrane stabilizer, the final product exhibited the best EGCG encapsulation effect.

[0181] Comparing Examples 1 and 13 to 15, it can be seen that the amount of rigid membrane stabilizer added affected the particle size, encapsulation yield, and storage stability of the final product. In Examples 1 and 13 to 15, the mass percentage of rigid membrane stabilizer was 0.15%, 0.1%, 0.2%, and 0.3%, respectively. The results show that with increasing amounts of rigid membrane stabilizer, the particle size of the product gradually increased, while the encapsulation yield initially increased and then decreased. When the mass percentage of rigid membrane stabilizer in the EGCG-based nanocomposition was 0.15% (Example 1), the encapsulation effect of EGCG was greatest.

[0182] Comparing Examples 1 and 16 to 18, it can be seen that the type of flexible membrane stabilizer affected the particle size, encapsulation yield, and storage stability of the final product. In Examples 1 and 16 to 18, the flexible membrane stabilizer was caprylic / capric triglyceride, caprylic / capric / linoleic triglyceride, linoleic acid, and octyldodecanol, respectively. The results show that when caprylic / capric triglyceride was chosen as the flexible membrane stabilizer, the final product exhibited the best encapsulation effect of EGCG.

[0183] Comparing Examples 1 and 19 to 21, it can be seen that the amount of added flexible membrane stabilizer affected the particle size and encapsulation yield of the final product. In Examples 1 and 19 to 21, the mass percentage of flexible membrane stabilizer was 1.5%, 0.5%, 1.0%, and 2.0%, respectively. The results show that with increasing amounts of added flexible membrane stabilizer, the particle size of the product decreased and then increased, while the encapsulation yield increased first and then decreased. When the mass percentage of flexible membrane stabilizer in the EGCG-based nanocomposition was 1.5% (Example 1), the encapsulation effect of EGCG was greatest.

[0184] By comparing examples 1, 22 and 23, it can be seen that because the quantities of precursors of the nano-composition added were different, the size of the The particle size, encapsulation yield, and storage stability of the ultimately prepared EGCG-based nanocompositions differed. In Examples 1, 22, and 23, the precursor mass percentages of the nanocomposition were 5%, 1%, and 10%, respectively. The results show that with increasing amounts of the nanocomposition precursor, the particle size of the product decreased and then increased, while the encapsulation yield initially increased and then decreased. When the precursor mass percentage of the nanocomposition in the EGCG-based nanocomposition was 5% (Example 1), the EGCG encapsulation effect was optimal.

[0185] By comparing Example 1, Comparative Example 1 and Comparative Example 2, it can be seen that, compared to traditional liposomes, the present application adopts a two-step self-assembly with phospholipids, consisting of first preparing a precursor of the nano-composition to encapsulate the EGCG and then performing a self-assembly on the precursor of the nano-composition with the phospholipids for hydrogenated protection, which ultimately results in an EGCG-based nano-composition with a reduced particle size, high encapsulation yield and best storage stability.

[0186] By comparing Example 1 and comparative examples 3 to 5, it can be seen that, compared to the use of a single rigid membrane stabilizer (comparative example 3) or the use of a single flexible membrane stabilizer (comparative example 4) or without any added membrane stabilizer (comparative example 5), in Example 1 of the present invention, the rigid membrane stabilizer and the flexible membrane stabilizer were used to adjust the composition of the phospholipid membranes, which ultimately makes it possible to obtain an EGCG-based nanocomposition with a reduced particle size, high encapsulation yield and best storage stability.

[0187] By comparing Example 1 with comparative examples 6 to 7, it can be seen that, during sample preparation, the amount of EGCG added affected the particle size, encapsulation yield, and storage stability of the final product. Compared to Example 6, in which EGCG was added in the second step of the phospholipid self-assembly of the EGCG-based nanocomposition, or to Example 7, in which EGCG was added after the preparation of the EGCG-based nanocomposition, in Example 1 of this application, the EGCG-based nanocomposition was prepared by first encapsulating EGCG to prepare a precursor of the nanocomposition and then performing a two-step phospholipid self-assembly, resulting in a final prepared sample with a reduced particle size, a high encapsulation yield, and improved storage stability.

[0188] Compared to Example 1 and Comparative Example 8 in which EGCG was encapsulated in a single step, in Example 1 of the present application, the EGCG-based nanocomposition was prepared by first encapsulating EGCG to prepare a precursor of the nanocomposition and performing self-assembly with phospholipids in two steps, which makes it possible to obtain a finally prepared sample having a reduced particle size, a high encapsulation yield and better storage stability, i.e. that the process of Example 1 of the present application has a better encapsulation effect of EGCG.

[0189] Compared to Example 1 and Comparative Example 9 in which EGCG was added to the aqueous phase and mixed with an oily phase to prepare a precursor of the nano-composition, in Example 1 of the present application, EGCG was added to an oily phase and mixed with an aqueous phase to prepare a precursor of the nano-composition, resulting in an EGCG-based nano-composition finally prepared with a reduced particle size, high encapsulation yield and improved storage stability.

[0190] Compared to Example 1, Comparative Example 10 and Comparative Example 11 in which the precursor of the nano-composition was modified with chitosan (Comparative Example 10) and gum arabic (Comparative Example 11), in Example 1 of the present application, hydrogenated lecithin was used to protect the precursor of the nano-composition, which ultimately makes it possible to obtain an EGCG-based nano-composition with a reduced particle size, a high encapsulation yield and the best storage stability.

[0191] Compared to Example 1 and Comparative Example 12 in which a form of nanoemulsion was adopted for the encapsulation of EGCG, in Example 1 of the present application, the EGCG-based nanocomposition was prepared by first encapsulating EGCG to prepare a precursor of the nanocomposition and performing a two-step self-assembly with phospholipids, which makes it possible to obtain a finally prepared sample exhibiting a reduced particle size, a high encapsulation yield and better storage stability.

[0192] Example of test 4 (test of vesicle deformation capacity)

[0193] Dispense 1 ml of EGCG-based nanocomposition into a syringe and insert the syringe into the cannula of an extruder mounted on a vertical support, with the needle pointing downwards and the piston segment in contact with a 50 N loading unit. Bring a probe into contact with the syringe piston at a constant speed of 1 mm / s and force the EGCG-based nanocomposition through a 50 nm polycarbonate membrane inside the extruder cannula. Pretreat the polycarbonate membrane with ultrapure water before each test.

[0194]

[0195]

[0196]

[0197]

[0198] The particle size of the EGCG-based nanocomposition was characterized before and after pressing using a Malvem Nano-ZS90 dynamic light scattering particle size analyzer. The test angle was 90° and the test temperature was 25°C. Three parallel tests were performed for each group, and arithmetic means were calculated from the test results. Calculate the rate of change in particle size of the EGCG-based nanocomposition before and after pressing using formula (2). A. - A. x 1 (Formula 2) "As where, DhO represents the particle size of the EGCG-based nanocomposition before pressing Dhe represents the particle size of the EGCG-based nanocomposition after pressing. Table 2: Variation in sample particle size before and after pressing [Tables 2] Samples Particle size before pressing (nm) Particle size after pressing (nm) Rate of change (%) Example 1 83.4 80.8 3.08 Example 2 105.2 94.3 10.40 Example 3 97.7 86.4 11.56 Example 4 126.2 108.2 14.32 Example 5 169.4 141.9 16.20 Example 6 88.4 79.0 10.68 Example 7 105.3 91.8 12.77 Example 8 134.2 116.6 13.11 Example 9 145.9 120.9 17.16 Example 10 102.9 95.1 7.54 Example 11 98.0 88.9 9.31 Example 12 106.9 96.1 10.10 Example 13 88.5 75.4 14.74 Example 14 107.0 93.2 12.86 Example 15 125.0 105.3 15.75 Example 16 98.8 87.8 11.09 Example 17 103.1 87.3 15.36 Example 18 105.1 87.4 16.89 Example 19 120.4 104.6 13.09 Example 20 105.1 93.0 11.49 Example 21 112.6 94.8 15.78 Example 22 97.0 85.8 11.47 Example 23 133.4 113.8 14.69 Example 24 134.0 105.1 21.55 Example 25 150.7 115.8 23.20 Comparative Example 1 164.5 95.9 41.70 Comparative Example 2 212.7 119.5 43.82 Comparative example 3 212.8 91.9 56.81 Comparative example 4 143.7 68.5 52.33 Comparative example 5 155.6 65.6 57.84 Comparative example 6 148.6 99.3 33.18 Comparative example 7 137.2 86.9 36.66 Comparative example 8 155.3 97.2 37.41 Comparative example 9 188.6 104.3 44.70 Comparative example 10 212.8 71.9 66.21 Comparative example 11 201.6 74.4 63.10 Comparative example 12 415.8 168.9 59.38

[0199] As shown in Table 2, comparing Examples 1 to 5, it can be seen that the amounts of soy lecithin added to the nanocomposition precursors differed, and that the deformation capacities of the ultimately prepared EGCG-based nanocompositions also differed. With increasing amounts of soy lecithin added to the nanocomposition precursor, the deformation capacity of the ultimately prepared EGCG-based nanocomposition initially increased and then decreased. When the mass percentage of soy lecithin in the nanocomposition precursor reached 5% (Example 1), the deformation capacity of the ultimately prepared EGCG-based nanocomposition was at its highest, and the rate of change in particle size of the sample before and after pressing was the smallest, namely 3.08%; therefore, the sample from Example 1 could effectively avoid the sudden release of EGCG due to the destruction of its structure, i.e., the EGCG-based nanocomposition from Example 1 exhibited better resistance to pressing and better stability.

[0200] Comparing Examples 1 and 6 to 9, it can be seen that because the amounts of hydrogenated lecithin added were different, the deformation capacity of the ultimately prepared EGCG-based nanocompositions was different.With increasing amounts of hydrogenated lecithin added, the deformation capacity of the finally prepared EGCG-based nanocomposition initially increased and then decreased; when the mass percentage of hydrogenated lecithin in the finally prepared EGCG-based nanocomposition reached 0.3% (Example 1), the deformation capacity of the finally prepared EGCG-based nanocomposition was at its best.

[0201] Comparing Examples 1 and 10 to 12, it can be seen that because the types of rigid membrane stabilizer differed, the deformation capacity of the ultimately prepared EGCG-based nanocompositions also differed. In Examples 1 and 10 to 12, the rigid membrane stabilizer was cholesterol, sodium cholate, resorcinol, and phenylalanine, respectively. The results show that when cholesterol was chosen as the rigid membrane stabilizer, the final EGCG-based nanocomposition exhibited the best deformation capacity, better compressive strength, and better stability.

[0202] By comparing examples 1 and 13 to 15, it can be seen that the amount of rigid membrane stabilizer added affected the deformation capacity of the final product. The results show that, with increasing amounts of rigid membrane stabilizer added, the deformation capacity of the finally prepared EGCG-based nanocomposition initially increased and then decreased. When the mass percentage of rigid membrane stabilizer in the EGCG-based nanocomposition reached 0.15% (example 1), the finally prepared EGCG-based nanocomposition exhibited the best deformation capacity, better pressing strength, and better stability.

[0203] By comparing examples 1 and 16 to 18, it can be seen that the type of rigid membrane stabilizer added affected the deformation capacity of the final product. The results show that when caprylic / capric triglyceride was chosen as the flexible membrane stabilizer (example 1), the final product exhibited the best deformation capacity, better compressive strength, and better stability.

[0204] By comparing examples 1 and 19 to 21, it can be seen that the amount of flexible membrane stabilizer added affected the deformation capacity of the final product. The results show that, with increasing amounts of stabilizer With the addition of a flexible membrane, the deformation capacity of the product initially increased and then decreased. When the mass percentage of flexible membrane stabilizer in the EGCG-based nanocomposition reached 1.5% (Example 1), the finally prepared EGCG-based nanocomposition exhibited the best deformation capacity, better resistance to pressing, and better stability.

[0205] By comparing Examples 1, 22, and 23, it can be seen that because the quantities of nanocomposition precursors added differed, the deformation capacities of the ultimately prepared EGCG-based nanocompositions also differed. The results show that, with increasing quantities of the nanocomposition precursor added, the deformation capacity of the product initially increased and then decreased. When the mass percentage of the nanocomposition precursor in the EGCG-based nanocomposition reached 5% (Example 1), the ultimately prepared EGCG-based nanocomposition exhibited the best deformation capacity, better compressive strength, and better stability.

[0206] By comparing example 1, comparative example 1 and comparative example 2, it can be seen that, compared to traditional liposomes, the present application adopts a two-step self-assembly with phospholipids, consisting of first preparing a precursor of the nano-composition to encapsulate the EGCG and then performing a self-assembly on the precursor of the nano-composition with the phospholipids for hydrogenated protection, which ultimately results in an EGCG-based nano-composition exhibiting the best deformation capacity, better resistance to pressing and better stability.

[0207] By comparing example 1 and comparative examples 3 to 5, it can be seen that, compared to the use of a single rigid membrane stabilizer (comparative example 3) or the use of a single flexible membrane stabilizer (comparative example 4) or without any added membrane stabilizer (comparative example 5), the present application uses the rigid membrane stabilizer and the flexible membrane stabilizer to adjust the composition of the phospholipid membranes, which ultimately makes it possible to obtain an EGCG-based nanocomposition with the best deformation capacity, better resistance to pressing and better stability.

[0208] By comparing Example 1 with comparative examples 6 and 7, it can be seen that, during sample preparation, the amount of EGCG added affected the deformation capacity of the final product. Compared to Example 6, in which EGCG was added in the second step of self-assembly with phospholipids in the preparation of the EGCG-based nanocomposition, or to Example 7, in which EGCG was added after the preparation of the EGCG-based nanocomposition, in Example 1 of this application, the EGCG-based nanocomposition was prepared by first encapsulating the EGCG to prepare a precursor. of the nano-composition and by performing a self-assembly with phospholipids in two steps, which makes it possible to obtain a finally prepared sample exhibiting the best deformation capacity, better resistance to pressing and better stability.

[0209] Compared to Example 1 and Comparative Example 8 in which EGCG was encapsulated in a single step, in Example 1 of the present application, the EGCG-based nanocomposition was prepared by first encapsulating EGCG to prepare a precursor of the nanocomposition and performing a self-assembly with phospholipids in two steps, which makes it possible to obtain a finally prepared sample exhibiting the best deformation capacity, better resistance to pressing and better stability.

[0210] Compared to Example 1 and Comparative Example 9 in which EGCG was added to the aqueous phase and mixed with an oily phase to prepare a precursor of the nano-composition, in Example 1 of the present application, EGCG was added to an oily phase and mixed with an aqueous phase to prepare a precursor of the nano-composition, which makes it possible to obtain a nano-composition based on the finally prepared EGCG exhibiting the best deformation capacity, better resistance to pressing and better stability.

[0211] Compared to Example 1, Comparative Example 10 and Comparative Example 11 in which the precursor of the nano-composition was modified with chitosan (Comparative Example 10) and gum arabic (Comparative Example 11), in Example 1 of the present application, hydrogenated lecithin was used to protect the precursor of the nano-composition, which ultimately makes it possible to obtain an EGCG-based nano-composition exhibiting the best deformation capacity, better resistance to pressing and better stability.

[0212] Example of test 5 (transdermal absorption test)

[0213] The transdermal absorption capacity (permeability) of the EGCG-based nanocomposition of Examples 1 to 21 and Comparative Examples 1 to 13 was evaluated through transdermal absorption (permeability) tests, comprising the following steps:

[0214] In in vitro transdermal absorption tests, a vertical diffusion pool was used and bare mouse skin was used as a model (abdominal skin, subcutaneous fat layer, and blood vessels were removed). PBS solution was used as the receiving solution. A piece of skin was fixed between the supply pool and the receiving pool, with the skin layer facing upwards, and equilibrated for 20 min. Samples were taken and added to the supply pool, and the receiving solutions were extracted after 1 h, 4 h, 8 h, and 24 h. The receiving solutions were demulsified by ultrasound with a mixed solution composed of methanol and The water was heated for 30 min, using a receiving solution ratio of methanol:water = 1:4:5 (v / v). The solution was then filtered through a 0.45 µm organic filter membrane. The EGCG content was determined using high-performance liquid chromatography (HPLC, Shimadzu, Japan), and the cumulative unit area penetration was calculated according to formula (3). Three parallel tests were performed for each group, and the arithmetic mean values ​​were calculated on the test results. •i >, C =__!___(Formula 3)

[0215] Where, 2n represents the cumulative unit area penetrability of the sample at time t (ug / cm2), A represents the penetration area, Cn represents the measured value of the EGCG concentration at time t, Q represents the measured value of the EGCG concentration sampled at time t, V represents the total volume of the receiving solution and Vo represents the sampling volume at each time point.

[0216] Table 3 Transdermal Test Results

[0217] [Tables3] Samples Cumulative Unit Area Penetration Quantity (g / cm²) Ih 4h 8h 24h Example 1 4.31 15.68 28.12 89.32 Example 2 3.5 12.4 23 73.4 Example 3 3.7 13.5 24.7 78.5 Example 4 3.4 12.1 22.1 71.4 Example 5 3.3 12.3 21.8 70.2 Example 6 3.6 13.6 24.1 76.3 Example 7 3.4 12.7 22.4 72.1 Example 8 3.5 12.5 22.2 72.7 Example 9 3.3 12.1 21.6 68.8 Example 10 3.9 14.5 25.3 80.1 Example 11 3.7 13.7 24.1 77.6 Example 12 3.6 13.6 24.0 75.4 Example 13 3.5 12.1 22.9 71.6 Example 14 3.5 12.8 22.7 72.6 Example 15 3.4 12.6 22.1 70.2 Example 16 3.5 12.7 23.0 73.1 Example 17 3.4 12.5 22.2 70.4 Example 18 3.3 12.1 21.9 69.7 Example 19 3.5 12.0 23.5 73.5 Example 20 3.5 13.1 23.6 74.2 Example 21 3.3 12.5 21.9 69.8 Comparative Example 1 1.9 7.1 12.6 42.5 Comparative Example 2 2.2 7.5 13.4 40.1 Comparative Example 3 1.7 6.3 11.2 35.4 Comparative Example 4 1.8 6.4 11.6 36.7 Comparative example 5 1.4 5.6 10.3 32.6 Comparative example 6 2.1 7.4 13.7 43.5 Comparative example 7 2.1 7.5 13.5 44.2 Comparative example 8 2.2 7.2 14.1 44.8 Comparative example 9 1.9 7.1 13.1 41.3 Comparative example 10 1.6 5.7 10.7 33.7 Comparative example 11 1.6 5.1 9.9 31.5 Comparative example 12 1.8 6.5 12.3 38.7 Comparative example 13 (Aqueous solution of EG CG) 0.45 2.67 5.18 14.71

[0218] As shown in Table 3, by comparing Examples 1 to 5, it can be seen that in the precursors of the nano-composition, the quantities of soy lecithin added were different, and that the transdermal capacity of the EGCG-based nano-compositions finally prepared were different. With increasing amounts of soy lecithin added to the nano-composition precursor, the transdermal capacity of the ultimately prepared EGCG-based nano-composition initially increased and then decreased. When the mass percentage of soy lecithin in the nano-composition precursor reached 5% (Example 1), the cumulative unit area penetration of EGCG in the final product at 1h, 4h, 8h, and 24h was 4.31 Jwg / cm², 115 Jwg / cm², 28.12 Jwg / cm², and 89.32 Jwg / cm², respectively. superior to that of the samples in examples 2 to 5, which showed that the transdermal capacity of example 1 was the best.

[0219] Comparing Examples 1 and 6 to 9, it can be seen that because the amounts of hydrogenated lecithin added differed, the transdermal capacity of the ultimately prepared EGCG-based nanocompositions also differed. In Example 1 of this application, the mass percentage of hydrogenated lecithin was 0.3%, and the cumulative unit area penetration of EGCG in the final product was the highest, demonstrating that the transdermal capacity of Example 1 was the best.

[0220] Comparing Examples 1 and 10 to 15, both the type and quantity of the added rigid membrane stabilizer affected the transdermal capacity of the final product. When cholesterol was chosen as the rigid membrane stabilizer at a mass percentage of 0.15% (Example 1), the cumulative unit area penetration of EGCG into the final product was highest, indicating that the corresponding transdermal capacity was best.

[0221] Comparing Examples 1 and 16 to 21, both the type and quantity of the added flexible membrane stabilizer affected the transdermal capacity of the final product. When caprylic / capric acid triglyceride was chosen as the rigid membrane stabilizer at a mass percentage of 1.5% (Example 1), the cumulative unit area penetration of EGCG into the final product was highest, indicating that the corresponding transdermal capacity was best.

[0222] By comparing example 1, comparative example 1 and comparative example 2, it can be seen that, compared to traditional liposomes, the present application adopts a two-step self-assembly with phospholipids, consisting of first preparing a precursor of the nano-composition to encapsulate the EGCG and then performing a self-assembly on the precursor of the nano-composition with the phospholipids for hydrogenated protection, which makes it possible to obtain a final product exhibiting the highest cumulative unit area penetration of EGCG, showing that the corresponding transdermal capacity was the best.

[0223] By comparing example 1 and comparative examples 3 to 5, it can be seen that, compared to the use of a single rigid membrane stabilizer (comparative example 3) or the use of a single flexible membrane stabilizer (comparative example 4) or without any added membrane stabilizer (comparative example 5), the present application uses both the rigid membrane stabilizer and the flexible membrane stabilizer to adjust the composition of the phospholipid membranes, resulting in a final product exhibiting the highest cumulative unit area penetration of EGCG, showing that the corresponding transdermal capacity was the best.

[0224] Comparing Example 1 and comparative examples 6 and 7, with respect to Example 6 in which EGCG is added in the second step of self-assembly with phospholipids in the preparation of the EGCG-based nanocomposition or with respect to Example 7 in which EGCG is added after the preparation of the EGCG-based nanocomposition, in Example 1 of the present application, the EGCG-based nanocomposition was prepared by first encapsulating EGCG to prepare a precursor of the nanocomposition and performing a two-step self-assembly with phospholipids, which makes it possible to obtain a final product exhibiting the highest cumulative unit area penetration of EGCG, showing that the corresponding transdermal capacity was the best.

[0225] Compared to Example 1 and Comparative Example 8 in which EGCG was encapsulated in a single step, in Example 1 of the present application, the EGCG-based nanocomposition was prepared by first encapsulating EGCG to prepare a precursor of the nanocomposition and performing a self-assembly with phospholipids in two steps, which makes it possible to obtain a final product exhibiting the highest cumulative unit area penetration of EGCG, showing that the corresponding transdermal capacity was the best.

[0226] Compared to Example 1 and Comparative Example 9 in which EGCG was added to the aqueous phase and mixed with an oily phase to prepare a precursor of the nano-composition, in Example 1 of the present application, EGCG was added to an oily phase and mixed with an aqueous phase to prepare a precursor of the nano-composition, resulting in a final product exhibiting the highest cumulative unit area penetration of EGCG, showing that the corresponding transdermal capacity was the best.

[0227] Compared to Example 1, Comparative Example 10 and Comparative Example 11 in which the precursor of the nano-composition was modified with chitosan (Comparative Example 10) and gum arabic (Comparative Example 11), in Example 1 of the present application, hydrogenated lecithin was used to protect the precursor of the nano-composition, resulting in a final product exhibiting the highest cumulative unit area penetration of EGCG, showing that the corresponding transdermal capacity was the best.

[0228] Compared to Example 1 and comparative examples 12 and 13, in which a form of nanoemulsion was adopted for the encapsulation of EGCG (comparative example 12) and an aqueous solution of EGCG was adopted (comparative example 13), in Example 1 of the present application, the EGCG-based nanocomposition was prepared by first encapsulating EGCG to prepare a precursor of the nanocomposition and by performing a two-step self-assembly with phospholipids, resulting in a final product exhibiting the cumulative penetration quantity the highest unit area of ​​EGCG, showing that the corresponding transdermal capacity was the best.

[0229] Example of test 6 (antioxidation test - inhibition of reactive oxygen species ROS)

[0230] Digest human skin keratinocytes (HaCaT), prepare a cell suspension and inoculate into a 96-well plate at 1.0x105 to 2.0x105 cells / well. Culture in DMEM medium for 24 h, change the medium, define a blank group (add PBS buffer, without light exposure), a control group (add PBS buffer, with light exposure) and a test group (add the samples from the examples and comparative examples with an addition of 10% of the amount of cell culture medium), and culture for 24 h, then expose the control group and the test group to UVA radiation (dose of 6 to 10 J / cm2). Remove the cell culture at the end of light exposure, wash 3 times with PBS, add 100 qL of DCFH-DA at 10 qmol / L to each well and incubate in a CO2 incubator for 20 min.Wash 3 times with PBS at the end of incubation, add serum-free DMEM medium, and determine the ROS content using a fluorescence microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The relative ROS level of the cells is the ratio of the OD value of the test group (or the OD value of the control group) to the OD value of the blank group.

[0231] Table 4: Relative ROS levels of HaCaT cells subjected to photo-damage and treated with the sample

[0232] [Tables4] Samples Relative ROS level of cells (%) Example 1 102.2 Example 2 111.5 Example 3 109.7 Example 4 112.8 Example 5 113.1 Example 6 109.2 Example 7 112.4 Example 8 113.7 Example 9 113.8 Example 10 107.5 Example 11 109.2 Example 12 110.6 Example 13 112.8 Example 14 111.1 Example 15 113.1 Example 16 110.9 Example 17 113.3 Example 18 112.4 Example 19 110.8 Example 20 110.4 Example 21 113.5 Comparative Example 1 121.3 Comparative Example 2 122.4 Comparative Example 3 125.1 Comparative Example 4 123.2 Comparative Example 5 123.7 Comparative Example 6 119.5 Comparative Example 7 120.2 Comparative Example 8 120.0 Comparative Example 9 123.8 Comparative Example 10 125.6 Comparative Example 11 125.5 Comparative Example 12 123.7 Example Comparative 13 (Aqueous solution of EGCG) 124.2 White group 100 Control group 152.3

[0233] Table 4 shows that the relative ROS level of HaCaT cells subjected to photo-damage and treated with the EGCG-based nanocomposition obtained in Examples 1 to 21 was lower than that of the control group, indicating a good antioxidant effect in reducing light damage. Moreover, the relative ROS level of HaCaT cells subjected to photo-damage and treated with the EGCG-based nano-composition obtained in examples 1 to 21 was lower than that of comparative examples 1 to 13, indicating a better antioxidant effect of examples 1 to 21, in which the relative ROS level of HaCaT cells subjected to photo-damage and treated with the EGCG-based nano-composition of example 1 was the lowest, i.e. 2.2%, indicating that the EGCG-based nano-composition of example 1 had the best antioxidant effect and could effectively reduce photo-damage.

[0234] Example of test 7 Repair of DNA damage 1' Anti-photoaging

[0235] (1) DNA double-strand break is considered the most serious lesion DNA, H2AX, the full name "H2A histone family member X," also called H2AX, is one of the variants of the chromosomal histone H2A. Once the cell's double-stranded DNA is cut, a phosphorylation modification occurs at H2AX, and phosphorylated H2AX is formed, or γ-H2AX. The level of γ-H2AX can clearly reflect the degree of DNA damage and repair, and has been widely used in research on DNA damage and apoptosis, becoming an important marker of DNA damage.

[0236] The present example of a DNA damage repair test uses a DNA damage detection kit (y-H2AX immunofluorescence, murine monoclonal antibody, green), which can be purchased directly from Biyuntian Biotechnology Co., Ltd. of Shanghai. Digest human skin keratinocytes (HaCaT), prepare a cell suspension, and inoculate into a 96-well plate at 1.0 x 10⁵ to 2.0 x 10⁵ cells / well. Culture in DMEM medium for 24 h, and expose a white control group and a test group to UVA radiation (dose of 6 to 10 J / cm²). Remove the cell culture at the end of the light exposure and wash 3 times with PBS. Collect the cells by centrifugation and wash once with PBS. Completely absorb the PBS and disperse the cells. Add the fixing solution provided with the kit, gently suspend the cells and fix for 10 min.Centrifuge, discard the fixation solution, add the wash solution provided with the kit, and wash once. Resuspend the cells with a small amount of wash solution and add the suspension dropwise onto a coverslip or slide to create a smear. Dry thoroughly, wash twice with the wash solution, add 1 ml of Timmunostaining blocking solution, and leave at room temperature for 15 minutes. Absorb the immunoblocking solution, add 1 ml of murine monoclonal antibody y-H2AX, and incubate at room temperature for 1 hour. Wash three times with the wash solution, add 1 ml of murine antibody 488, and incubate at room temperature for 1 hour. Wash. Wash the samples twice with the washing solution, add 1 ml of nuclear staining solution (DAPI), stain at room temperature for 5 min, absorb the nuclear staining solution, and wash three times with the washing solution. Finally, photograph the samples using confocal laser imaging and analyze them.

[0237] The results of DNA damage repair (y-H2AX method) of Example 1, Comparative Example 13 (EGCG aqueous solution), and vitamin C (VC) are shown in [Fig. 1]. The results show that the cells were exposed to light (ultraviolet radiation), which causes DNA damage, and exhibited significantly reduced green fluorescence content after treatment with the test groups of Example 1 and Comparative Example 13, and lower than that of the VC group, indicating that the repair effect of the test group (Example 1 and Comparative Example 13) on DNA-damaged cells was better than that of the VC control group.Furthermore, after treatment of example 1, the green fluorescence content of the cells was significantly lower than that of comparator example 13, indicating that the EGCG-based nanocomposition in example 1 had a better repair effect on DNA-damaged cells than the aqueous EGCG solution without wrapping treatment.

[0238] Example of test 8 Inhibition of protein carbonylation - Skin anti-yellowing effect test

[0239] Protein carbonylation is an irreversible non-enzymatic carbonylation modification of proteins and the pathways of formation of carbonylated proteins are grouped into two types depending on the participation of ROS or not: (1) ROS directly oxidize the amino acids of the side chain of proteins to form carbonylated proteins (i.e. free radical oxidation); (2) Proteins are transformed into carbonylated proteins after the processes of lipid oxidation and non-enzymatic glycosylation (i.e. glycosylation).

[0240] Index detection principle: DNPH staining: The carbonyl content of proteins is a sensitive index of oxidative protein damage. 2,4-Dinitrophenylhydrazine (DNPH) colorimetry is the classic method for determining the carbonyl content of proteins. The carbonyl content of oxidized proteins increases, and the carbonyl can react with 2,4-dinitrophenylhydrazide to form 2,4-dinitrophenylhydrazone, which exhibits green fluorescence after fluorescence staining. The anti-carbonylation capacity of the sample can be assessed by analyzing the fluorescence intensity of the sample being tested; the lower the green fluorescence intensity, the fewer carbonyl products are present.

[0241] The carbonyl group of proteins is an early marker of a variety of amino acids in the process of protein oxidation modification, and its content indicates the degree of damage due to protein oxidation, which is the main indicator for To assess protein oxidation damage, inoculate L929 cells in logarithmic growth phase into a 6-well plate (100,000 cells / well), expose the wells to ultraviolet light for 10 minutes per well once the cells have adhered to the well walls, administer different samples at the same concentration to different wells, and treat the cells 48 hours after administration. The carbonyl group reacts with 2,4-dinitrophenylhydrazine to form red 2,4-dinitrophenylhydrazone with a characteristic absorption peak at 370 nm.

[0242] Cells exposed to radiation for 10 min without sample administration constituted the positive control group; normal cells in logarithmic growth phase and cells without ultraviolet radiation exposure and without sample administration constituted the negative control groups. The samples added to the test group are the samples from Example 1, Comparative Example 13 (EGCG aqueous solution), and vitamin C (VC), and the results of the protein carbonylation inhibition assay are shown in [Fig. 2].The results show that exposure of cells (to ultraviolet light) caused a significant increase in protein carbonyl content, the protein carbonyl content significantly decreased after treatment with the test group of example 1 and the test group of comparative example 13, which was lower than that of the VC group, indicating that the protein carbonylation inhibition effect of example 1 and comparative example 13 was better than that of the VC test group, i.e. EGCG had a better protein carbonylation inhibition effect than VC.Furthermore, after treatment with the test group in Example 1, the carbonyl content of the proteins was significantly lower than that of the test group in Comparative Example 13, indicating that the EGCG-based nanocomposition in Example 1 had a better protein carbonylation inhibition effect than an untreated aqueous EGCG solution. Thus, the EGCG-based nanocomposition of the present invention can play an antioxidant, anti-photoaging, and anti-yellowing role in the skin.

Claims

1. Demands A process for preparing a nano-composition based on EGCG with powerful antioxidant and anti-photoaging effects, characterized in that it comprises the following steps: (1) Preparation of a precursor of the nano-composition: Add a phospholipid and EGCG to an organic solvent and stir uniformly to obtain an organic phase; add a polyol to water and stir uniformly to obtain an aqueous phase; add the organic phase dropwise to the aqueous phase at a constant rate, and once the dropwise addition is complete, stir again to obtain a mixed solution; remove the organic solvent from the mixed solution; and perform a drying treatment to obtain a precursor of the nano-composition; Expressed as a mass percentage, the phospholipid content in the mixed solution is 1% to 10%; (2) Preparation of a nano-composition with a double phospholipid: Add a phospholipid, a rigid membrane stabilizer and a flexible membrane stabilizer to the polyol, and stir uniformly to obtain an alcoholic phase; add the precursor of the nano-composition to water, stir and disperse until a homogeneous dispersion is obtained to obtain an aqueous phase; add the aqueous phase dropwise to the alcoholic phase at a constant rate, and once the dropwise addition is complete, stir again to obtain a mixed solution; and perform a nano-treatment on the mixed solution to obtain an EGCG-based nano-composition, the EGCG representing a mass percentage of 0.6% to 6.0% of the EGCG-based nano-composition; Expressed as a mass percentage, the phospholipid content in the mixed solution is 0.1% to 1.0%, the rigid membrane stabilizer content is 0.1% to 0.3%, and the flexible membrane stabilizer content is 0.5% to 2.0%; The molecular structure of said rigid membrane stabilizer comprises a polycyclic structure unit or a benzene ring structure unit; the molecular structure of said flexible membrane stabilizer is a linear chain structure or a linear chain compound.

2. A preparation method according to claim 1, characterized in that the rigid membrane stabilizer comprises one or more of cholesterol, phytosterol, sodium cholate, vitamin D, resorcinol, phenylalanine and phenylpropanol; the flexible membrane stabilizer comprises one or more of caprylic / capric acid triglyceride, caprylic / capric / linoleic acid triglyceride, caprylic / capric / uric acid triglyceride, oleic acid, linoleic acid, octyldodecanol and octyldodecyl oleate.

3. A preparation method according to claim 1 or 2, characterized in that EGCG represents a mass percentage of 1% to 5% of the EGCG-based nanocomposition.

4. A preparation process according to claim 3, characterized in that, expressed as mass percentages, - at step (1), the phospholipid content in the mixed solution is 3% to 7%, the EGCG content is 10% to 20%, the polyol content is 1% to 10%, the organic solvent content is 10% to 50% and the water content is 13% to 78%; - at step (2), the phospholipid content in the mixed solution is 0.1% to 0.5%, the rigid membrane stabilizer is 0.1% to 0.2%, the flexible membrane stabilizer content is 1% to 2%, the polyol content is 5% to 50%, the precursor content of the nano-composition is 1.0% to 10% and the water content is 37.3% to 88.4%.

5. A preparation method according to claim 4, characterized in that, the phospholipid in step (1) is at least one of soy lecithin, hydrogenated soy lecithin, enzymatically hydrolyzed lecithin, dipalmitoylphosphatidylcholine, palmitoylphosphatidylglycerol and egg yolk lecithin; the phospholipid in step (2) is at least one of soy lecithin, hydrogenated soy lecithin, dipalmitoylphosphatidylcholine, palmitoylphosphatidylglycerol and egg yolk lecithin.

6. A preparation process according to claim 5, characterized in that the organic solvent in step (1) is one or more of absolute ethanol, methanol, dichloromethane, trichloromethane, acetone, tetrahydrofuran, n-hexane, petroleum ether and ethyl acetate; - the polyol in step (1) is a sugar alcohol, which is one or more of maltitol, D-sorbitol, xylitol, erythritol, mannitol and lactitol; - the polyol in step (2) is at least one of the following: glycerol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,2-hexanediol, ethoxydiethylene glycol, dipropylene glycol and polyethylene glycol.

7. A preparation method according to claim 6, characterized in that, - the conditions of the first two stirrings in step (1): the stirring speed is 100 to 800 rpm; the stirring temperature is 30 to 80°C; the stirring time is 20 to 60 min; and the drop-by-drop addition rate is 1 to 10 ml / min; the conditions of the third stirring: the stirring speed is 100 to 800 rpm; the stirring temperature is 30 to 80°C; the stirring time is 10 to 30 min; the organic solvent is removed from the mixed solution by rotary evaporation at a rotary evaporation speed of 100 to 350 rpm; the rotary evaporation temperature is 30 to 80°C; and the rotary evaporation time is 1 to 3 h; - the conditions of the first two stirrings in step (2): the stirring speed is 100 to 800 rpm; the stirring temperature is 10 to 50°C; the stirring time is 20 to 60 minutes; and the drop-by-drop addition rate is 1 to 10 ml / min;The conditions for the third stirring: the stirring speed is 100 to 800 rpm; the stirring temperature is 10 to 50°C; and the stirring time is 10 to 30 minutes.

8. A preparation method according to claim 7, characterized in that the nano-treatment in step (2) comprises high-speed shear dispersion and / or microfluid homogenization; the rotation speed of the high-speed shear dispersion is 6000 to 12000 rpm and the shear dispersion time is 10 to 30 min; a homogenization pressure of the microfluid homogenization is 50 to 120 MPa and a number of homogenization cycles is 2 to 6.

9. Nano-composition based on EGCG prepared by the preparation process according to any one of claims 1 to 8.

10. Applications of the EGCG-based nanocomposition according to claim 9 in topical dermal formulations.