Application of ground tea leaf extract in preparation of skin care product composition

Abstract

The invention provides application of a ground tea leaf extract in preparation of a skin care product composition, and belongs to the technical field of skin care product manufacturing. The skincare product extract provided by the invention is prepared from the ground tea leaf extract, and by virtue of effective components in the ground tea leaf extract, the expression of iNOS and TRPV1 can be synchronously and effectively inhibited, so that the skincare product extract and the corresponding skincare product have the effects of resisting inflammation, relieving allergy, resisting wrinkles and the like.

Description

Technical Field

[0001] The present application relates to the technical field of skin care product manufacturing, and in particular, to an application of tea extract in the preparation of a skin care product composition. Background Art

[0002] Cytokine-induced nitric oxide synthase (iNOS) and capsaicin receptor (TRPV1) are closely related to human inflammatory response, cell aging and decreased skin elasticity. The iNOS pathway is as follows: proinflammatory factors induce a large amount of iNOS expression, iNOS reacts with L-arginine to generate high concentrations of nitric oxide (NO), which can cause skin inflammation; at the same time, NO, as a free radical, can cause metabolic hypoxia in cells, further causing the destruction of mitochondrial oxidative phosphorylation function, causing mitochondria to release up to ten times more reactive oxygen species (ROS). ROS directly enter the cell nucleus through the cytoplasm and nuclear pores, and can also cause cell aging. The TRPV1 pathway is as follows: TRPV1 on the nociceptor is stimulated by proinflammatory factors, and the cation channel opens, causing skin sensitivity, redness, itching and pain, etc.; in addition, high temperature and ultraviolet light can also increase the expression of TRPV1, causing calcium ion influx, inducing the expression of matrix metalloproteinase 1, and leading to decreased skin elasticity. In the field of skin care products, how to simultaneously inhibit the expression of iNOS and TRPV1 through skin care products is a major problem currently faced. Summary of the invention

[0003] The purpose of the present application is to provide an application of a tea leaf extract in the preparation of a skin care composition. The skin care extract is prepared from the tea leaf extract. With the help of the active ingredients in the tea leaf extract, the expression of iNOS and TRPV1 can be synchronously and effectively inhibited, thereby making the skin care extract have anti-inflammatory, soothing and anti-wrinkle effects.

[0004] The embodiment of the present application is implemented as follows:

[0005] In a first aspect, an embodiment of the present application provides an application of a tea extract in the preparation of a skin care composition having the efficacy of inhibiting the expression of cytokine-inducible nitric oxide synthase and capsaicin receptor.

[0006] In the above technical scheme, the tea extract contains a variety of ingredients that are beneficial to the human body, such as snow tea, scaly acid, horny acid, usnic acid, D-arabitol and mannitol, etc. The inventors found through molecular simulation docking that scaly acid and snow tea have high affinity with cytokine-inducible nitric oxide synthase (iNOS) and capsaicin receptor (TRPV1), respectively. Based on this, the tea extract is prepared into a skin care product composition, and with the help of the active ingredients in the tea extract, the expression of iNOS and TRPV1 can be synchronously and effectively inhibited, so that the skin care product extract has anti-inflammatory, soothing and anti-wrinkle effects.

[0007] In some optional embodiments, the preparation steps of the tea leaf extract include: extracting the tea leaf powder by water extraction to obtain an extract; filtering the extract to obtain outer membrane vesicles and filtrate respectively; flushing the outer membrane vesicles at the filter element with water and collecting them to obtain an outer membrane vesicle concentrate; mixing the outer membrane vesicle concentrate, filtrate and water and letting them stand to obtain the tea leaf extract.

[0008] In the above technical scheme, an outer membrane vesicle concentrate and a filtrate are first prepared according to the above process, and then the outer membrane vesicle concentrate, the filtrate and water are mixed and allowed to stand for a standing treatment, so that the various effective ingredients in the filtrate can enter the outer membrane vesicles along with the water during the standing process, that is, the outer membrane vesicles can act as a carrier and protective layer for the effective ingredients, thereby improving the stability and activity of the effective ingredients, so that the scaly acid and snow tea in the prepared tea extract can better bind to cytokine-inducible nitric oxide synthase (iNOS) and capsaicin receptor (TRPV1), thereby more effectively inhibiting the expression of iNOS and TRPV1.

[0009] In some optional embodiments, the step of filtering the extract includes: performing primary filtration on the extract using 5C filter paper to obtain a primary filtrate; performing secondary filtration on the primary filtrate using a microfiltration membrane to obtain a secondary filtrate; performing tertiary filtration on the secondary filtrate using an ultrafiltration membrane to obtain outer membrane vesicles and a filtrate, respectively.

[0010] In the above technical scheme, the first-stage filtration using 5C filter paper is mainly used to remove insoluble substances, the second-stage filtration using microfiltration membrane is mainly used to remove microparticles, bacteria, colloids and other substances, and the third-stage filtration using ultrafiltration membrane is to separate the outer membrane vesicles and filtrate. The three-stage filtration in sequence can obtain outer membrane vesicles and filtrate with higher purity, and also has the advantage of higher yield.

[0011] In some optional embodiments, in the three-stage filtration step, the ultrafiltration membrane used is an external pressure ultrafiltration membrane.

[0012] In the above technical solution, an external pressure ultrafiltration membrane is used in the three-stage filtration step, that is, the secondary filtrate is separated by an outflow method, which has the advantages of not easily damaging the outer membrane vesicles and having a higher separation efficiency.

[0013] In some optional embodiments, in the step of tertiary filtration, the filtration pressure is 0.1-0.6 MPa.

[0014] In the above technical solution, the filtration pressure in the tertiary filtration step is limited within the above range, which can achieve efficient separation of outer membrane vesicles and solution and also reduce the risk of outer membrane vesicles being damaged.

[0015] In some optional embodiments, the volume ratio of the outer membrane vesicle concentrate to the secondary filtrate is 1:(0.2-0.5).

[0016] In the above technical solution, the volume ratio of the outer membrane vesicle concentrate to the secondary filtrate is limited to the above range, so that the outer membrane vesicle concentrate has a relatively suitable concentration of outer membrane vesicles, thereby facilitating the subsequent preparation of tea extract as a raw material.

[0017] In some optional embodiments, before the step of mixing the outer membrane vesicle concentrate, filtrate and water and placing them aside for a static treatment, the step of concentrating the filtrate is also included so that the mass concentration of dry matter in the filtrate is 8-12 mg / mL.

[0018] In the above technical scheme, the filtrate is first concentrated to a mass concentration of dry matter within the above range, and then the tea leaf extract is prepared, so that the concentration of the active ingredients contained in the filtrate of different batches is basically the same, and then the content of the active ingredients in the tea leaf extracts of different batches is basically the same, which is convenient for subsequent mass production.

[0019] In some optional embodiments, the step of mixing the outer membrane vesicle concentrate, filtrate and water and then letting it stand comprises: mixing 1-5% of the outer membrane vesicle concentrate, 0.5-2% of the filtrate and the remainder of water in mass percentage and then letting it stand.

[0020] In the above technical solution, the outer membrane vesicle concentrate, filtrate and water are mixed according to the above mass proportions and then left to stand, so that the effective ingredients can enter the outer membrane vesicles more easily and fully.

[0021] In some optional embodiments, in the step of standing treatment, the standing time is 2 to 4 hours.

[0022] In the above technical solution, the duration of the static treatment is limited within the above range so that the effective ingredients can enter the outer membrane vesicles more fully.

[0023] In some optional embodiments, the skin care composition includes, by weight: 0.5-8 parts of tea leaf extract, 2-10 parts of polysaccharide-modified liposomes, 0.1-1 parts of Ganoderma lucidum extract, 5-10 parts of stabilizer and 5-10 parts of preservative.

[0024] In the above technical scheme, the skin care composition also contains polysaccharide-modified liposomes, which can fuse with the outer membrane vesicles in the tea leaf extract. On the one hand, it can increase the drug loading capacity of the outer membrane vesicles and help improve the stability and activity of the effective ingredients; on the other hand, by utilizing the principle that liposomes can carry water-soluble and fat-soluble components and deliver them to the deep skin, the utilization rate of the tea leaf extract can be improved.

[0025] In some alternative embodiments, the skin care product composition is used to prepare a skin care product.

[0026] In the above technical solution, the skin care composition is used to prepare skin care products. Since the skin care composition can synchronously and effectively inhibit the expression of iNOS and TRPV1, the corresponding skin care products also have anti-inflammatory, soothing and anti-wrinkle effects.

[0027] In some optional embodiments, the skin care product composition accounts for 0.5-1% by weight in the skin care product.

[0028] In the above technical solution, the mass proportion of the skin care product composition in the skin care product is limited to the above range, which can give the skin care product better stability and safety while taking into account better anti-inflammatory, soothing and anti-wrinkle effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 A process flow chart of a method for preparing a tea extract provided in an embodiment of the present application;

[0031] Figure 2 Physical pictures of erythema of a series of test samples provided for this application;

[0032] Figure 3 Actual pictures of wrinkles at different times for the control group provided for this application;

[0033] Figure 4 Actual pictures of wrinkles in the experimental group provided for this application at different times. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0035] It should be noted that “and / or” in this application, such as “Feature 1 and / or Feature 2”, refers to the three situations of “Feature 1” alone, “Feature 2” alone, or “Feature 1” plus “Feature 2”.

[0036] In addition, in the description of the present application, unless otherwise specified, the meaning of "multiple" in "one or more" refers to two or more; the range of "value a ~ value b" includes the two end values ​​"a" and "b", and the "unit" in "value a ~ value b + unit of measurement" represents the "unit of measurement" of both "value a" and "value b".

[0037] The following is a detailed description of the use of a tea extract in the preparation of a skin care composition according to an embodiment of the present application.

[0038] In a first aspect, an embodiment of the present application provides an application of a tea extract in the preparation of a skin care composition having the efficacy of inhibiting the expression of cytokine-inducible nitric oxide synthase and capsaicin receptor.

[0039] In the present application, the tea extract contains a variety of ingredients that are beneficial to the human body, such as snow tea, scaly acid, horny acid, usnic acid, D-arabitol and mannitol, etc. The inventors found through molecular simulation docking that scaly acid and snow tea have high affinity with cytokine-induced nitric oxide synthase (iNOS) and capsaicin receptor (TRPV1), respectively. Based on this, the tea extract is prepared into a skin care product composition, and with the help of the active ingredients in the tea extract, the expression of iNOS and TRPV1 can be synchronously and effectively inhibited, so that the skin care product extract has anti-inflammatory, soothing and anti-wrinkle effects.

[0040] As an example, the preparation steps of tea leaf extract include: extracting tea leaf powder by water extraction to obtain an extract; filtering the extract to obtain outer membrane vesicles and filtrate respectively; flushing the outer membrane vesicles at the filter element with water and collecting them to obtain an outer membrane vesicle concentrate; mixing the outer membrane vesicle concentrate, filtrate and water and letting them stand to obtain the tea leaf extract.

[0041] In this embodiment, an outer membrane vesicle concentrate and a filtrate are first prepared according to the above process, and then the outer membrane vesicle concentrate, the filtrate and water are mixed and allowed to stand for a standing treatment, so that the various active ingredients in the filtrate can enter the outer membrane vesicles along with the water during the standing process, that is, the outer membrane vesicles can act as a carrier and protective layer for the active ingredients, thereby improving the stability and activity of the active ingredients, so that the scaly acid and snow tea in the prepared tea extract can better bind to cytokine-inducible nitric oxide synthase (iNOS) and capsaicin receptor (TRPV1), thereby more effectively inhibiting the expression of iNOS and TRPV1.

[0042] It should be emphasized that in the field of skin care product manufacturing technology, at this stage, the aqueous solution after water extraction is usually directly used as a tea extract, and then used in the preparation of skin care product compositions; in this application, the inventor innovatively filtered the outer membrane vesicles during the water extraction process and used them as a carrier and protective layer for the active ingredients. Compared with directly using the aqueous solution after water extraction as the tea extract, it is found that the former can improve the stability and activity of the active ingredients and thus better exert the effects of each active ingredient. At the same time, how to efficiently obtain high-purity and relatively complete outer membrane vesicles in the water extraction process and how to make the active ingredients in the aqueous solution easily and thoroughly enter the outer membrane vesicles are also key points to be considered in this application.

[0043] It should be noted that the water extraction method is not limited and can be carried out according to conventional methods in the art.

[0044] As an example, the steps of water extraction include: mixing tea powder and water in a volume ratio of 1:(10-25), and then stirring the mixture at 35-45° C. for 2-5 hours to obtain an extract.

[0045] As an example, the steps of filtering the extract include: using 5C filter paper to perform primary filtration on the extract to obtain a primary filtrate; using a microfiltration membrane to perform secondary filtration on the primary filtrate to obtain a secondary filtrate; using an ultrafiltration membrane to perform tertiary filtration on the secondary filtrate to obtain outer membrane vesicles and filtrate, respectively.

[0046] In this embodiment, the primary filtration using 5C filter paper is mainly used to remove insoluble substances, the secondary filtration using microfiltration membrane is mainly used to remove microparticles, bacteria, colloids and other substances, and the tertiary filtration using ultrafiltration membrane is to separate the outer membrane vesicles and filtrate. The three-stage filtration in sequence can obtain outer membrane vesicles and filtrate with higher purity and also has the advantage of higher yield.

[0047] As an example, 5C filter paper is ADVANTEC quantitative filter paper with a pore size of 110 mm.

[0048] As an example, in the step of secondary filtration using a microfiltration membrane, the pore size of the microfiltration membrane is 0.22-0.45 μm, for example but not limited to the pore size of any one of 0.22 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, and 0.45 μm, or a range between any two of them; the filtration pressure is 0.1-0.6 MPa, for example but not limited to the filtration pressure of any one of 0.1 MPa, 0.2 MPa, 0.3 MPa, 0.4 MPa, 0.5 MPa and 0.6 MPa, or a range between any two of them; the material of the microfiltration membrane is selected from at least one of PVDF, PP and PET.

[0049] As an example, the pore size of the microfiltration membrane is 0.22 μm, the material of the microfiltration membrane is PVDF, and the filtration pressure is 0.1-0.3 MPa (for example, but not limited to, the filtration pressure is any one of 0.1 MPa, 0.15 MPa, 0.2 MPa, 0.25 MPa and 0.3 MPa or a range between any two).

[0050] As an example, in the step of three-stage filtration using an ultrafiltration membrane, the pore size of the ultrafiltration membrane is 40 to 50 nm, for example but not limited to the pore size of any one point value among 40 nm, 42 nm, 44 nm, 46 nm, 48 nm, and 50 nm, or a range value between any two of them; the material of the ultrafiltration membrane is selected from at least one of PVDF, PP and PET.

[0051] As an example, the pore size of the ultrafiltration membrane is 40 nm and the material of the ultrafiltration membrane is PVDF.

[0052] As an example, in the three-stage filtration step, the ultrafiltration membrane used is an external pressure ultrafiltration membrane.

[0053] In this embodiment, an external pressure ultrafiltration membrane is used in the three-stage filtration step, that is, the secondary filtrate is separated by an outflow method, which has the advantages of not easily damaging the outer membrane vesicles and having a higher separation efficiency.

[0054] In other possible implementations, an internal pressure ultrafiltration membrane may be used, that is, an internal flow method is used to separate the secondary filtrate.

[0055] As an example, in the three-stage filtration step, the filtration pressure is 0.1-0.6 MPa, for example but not limited to, the filtration pressure is any point value among 0.1 MPa, 0.2 MPa, 0.3 MPa, 0.4 MPa, 0.5 MPa and 0.6 MPa or a range value between any two of them.

[0056] In this embodiment, the filtration pressure in the tertiary filtration step is limited to the above range, which can achieve efficient separation of outer membrane vesicles and solution and also reduce the risk of outer membrane vesicle damage.

[0057] As an example, in the three-stage filtration step, the filtration pressure is 0.1-0.3 MPa, for example but not limited to, the filtration pressure is any one of 0.1 MPa, 0.15 MPa, 0.2 MPa, 0.25 MPa and 0.3 MPa or a range between any two of them.

[0058] As an example, the volume ratio of the outer membrane vesicle concentrate to the secondary filtrate is 1:(0.2-0.5), for example but not limited to any one of 1:0.2, 1:0.3, 1:0.4 and 1:0.5 or a range between any two of the volume ratios.

[0059] In this embodiment, the volume ratio of the outer membrane vesicle concentrate to the secondary filtrate is limited within the above range, so that the outer membrane vesicle concentrate has a relatively suitable concentration of outer membrane vesicles, thereby facilitating the subsequent preparation of tea extract as a raw material.

[0060] As an example, before the step of mixing the outer membrane vesicle concentrate, filtrate and water and letting them stand, the step of concentrating the filtrate is also included so that the mass concentration of dry matter in the filtrate is 8 to 12 mg / mL, for example but not limited to the mass concentration of any one of 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL and 12 mg / mL or a range between any two of them.

[0061] In this embodiment, the filtrate is first concentrated to a mass concentration of dry matter within the above range, and then the tea leaf extract is prepared, so that the concentration of the active ingredients contained in the filtrate of different batches is basically the same, and then the content of the active ingredients in the tea leaf extracts of different batches is basically the same, which is convenient for subsequent mass production.

[0062] It should be noted that the concentration treatment method is not limited and can be performed according to conventional methods in the art.

[0063] As an example, the filtrate is concentrated by rotary evaporation, wherein the processing temperature is 60° C., the rotation speed is 0-75 rpm, and the processing pressure is 80-100 mbar.

[0064] As an example, the step of mixing the outer membrane vesicle concentrate, filtrate and water and then letting it stand includes: mixing 1-5% of the outer membrane vesicle concentrate, 0.5-2% of the filtrate and the rest of the water in percentage by mass and then letting it stand.

[0065] In this embodiment, the outer membrane vesicle concentrate, the filtrate (ie, the filtrate after the concentration treatment) and water are mixed according to the above-mentioned mass ratio and then allowed to stand, so that the effective ingredients can enter the outer membrane vesicles more easily and fully.

[0066] As an example, in the step of static treatment, the static time is 2 to 4 hours, for example but not limited to, the static time is any one of 2 hours, 3 hours and 4 hours or a range between any two of them.

[0067] In this embodiment, the duration of the static treatment is limited to the above range so that the effective ingredients can enter the outer membrane vesicles more fully.

[0068] As an example, the preparation process of tea extract can be seen in Figure 1 .

[0069] As an example, the skin care composition includes, by weight: 0.5-8 parts of tea leaf extract, 2-10 parts of polysaccharide-modified liposomes, 0.1-1 parts of Ganoderma lucidum extract, 5-10 parts of stabilizer and 5-10 parts of preservative.

[0070] In this embodiment, the skin care composition also contains polysaccharide modified liposomes, which can fuse with the outer membrane vesicles in the tea leaf extract. On the one hand, the drug loading capacity of the outer membrane vesicles can be increased and the stability and activity of the effective ingredients can be helped to be improved; on the other hand, the principle that liposomes can carry water-soluble and fat-soluble components and deliver them to the deep skin can be utilized to improve the utilization rate of the tea leaf extract.

[0071] It should be noted that, except for the tea extract, the other components in the skin care product extract can be arranged according to the conventional selection in the art.

[0072] As an example, in the polysaccharide-modified liposome, the polysaccharide is selected from at least one of chitosan, pectin, hyaluronic acid and gum arabic.

[0073] As an example, the steps for preparing polysaccharide-modified liposomes include:

[0074] Lecithin is dissolved in anhydrous ethanol to obtain a mixed solvent; then the anhydrous ethanol in the mixed solvent is removed until a transparent lipid film is formed; then a sterile phosphate buffer solution (PBS) with a pH of 7.0 is added to the lipid film and the mixture is rotated and hydrated in a 50°C water bath at normal pressure to fully dissolve the lipid film in the PBS solution to obtain multilamellar liposomes; then the multilamellar liposomes are ultrasonically treated in an ice-water bath for 2 minutes and then placed in a 50°C water bath for 2 hours to obtain liposomes.

[0075] Weigh water-soluble chitosan (in terms of mass percentage, the mass ratio of water-soluble chitosan to liposome is 0.001:1) and dissolve it in sterile water, and stir to obtain a chitosan solution; stir and mix the chitosan solution and the above liposome solution to obtain polysaccharide-modified liposomes.

[0076] As an example, the stabilizer is selected from at least one of glycerol, 1,2-hexanediol, pentylene glycol and caprylic / capric / lauric triglyceride.

[0077] As an example, the preservative is selected from 1,3-butanediol.

[0078] As an example, the preparation steps of the skin care composition include: mixing tea leaf extract, polysaccharide-modified liposomes, red ganoderma extract, stabilizer and preservative, and then ultrasonically treating at room temperature for 2 minutes and homogenizing at 2000 rpm for 3 minutes to obtain the skin care composition.

[0079] As an example, the skin care product composition is used to prepare a skin care product.

[0080] In this embodiment, the skin care product composition is used to prepare skin care products. Since the skin care product composition can synchronously and effectively inhibit the expression of iNOS and TRPV1, the corresponding skin care products also have anti-inflammatory, soothing and anti-wrinkle effects.

[0081] As an example, the mass proportion of the skin care composition in the skin care product is 0.5-1%, for example but not limited to any one of 0.5%, 0.6%, 0.7%, 0.8%, 0.9% and 1% or a range between any two of them.

[0082] In this embodiment, the mass proportion of the skin care product composition in the skin care product is limited to the above range, which can give the skin care product better stability and safety while taking into account better anti-inflammatory, soothing and anti-wrinkle effects.

[0083] It should be noted that the types of skin care products are not limited and can be adaptively adjusted according to actual needs. For example, the skin care products can be essence, eye cream or face cream.

[0084] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0085] Example 1

[0086] The present invention provides a method for preparing a skin care composition, comprising the following steps:

[0087] Preparation of S1 tea extract

[0088] The ground tea leaves are washed, dried and then crushed, and the ground tea leaf powder is mixed with water in a volume ratio of 1:10, and then the mixture is stirred and extracted at 40° C. for 3 hours to obtain an extract.

[0089] The extract was filtered using 5C filter paper (ADVANTEC quantitative filter paper) with a pore size of 110 mm to obtain a primary filtrate; the primary filtrate was filtered using a microfiltration membrane, wherein the pore size of the microfiltration membrane was 22 μm, the material of the microfiltration membrane was PVDF, and the filtration pressure was 0.2 MPa, to obtain a secondary filtrate; the secondary filtrate was filtered using an ultrafiltration membrane, wherein the pore size of the ultrafiltration membrane was 40 nm, the material of the ultrafiltration membrane was PVDF, and the filtration pressure was 0.2 MPa, to obtain outer membrane vesicles and filtrate, respectively.

[0090] The outer membrane vesicles at the filter element were rinsed with water and collected to obtain an outer membrane vesicle concentrate, wherein the volume ratio of the outer membrane vesicle concentrate to the secondary filtrate was 1:0.2; the filtrate was concentrated using a rotary evaporator, wherein the treatment temperature was 60°C, the rotation speed was 72 rpm, and the treatment pressure was 80 mbar, until the filtrate volume was 30% of the initial volume; the filtrate was then centrifuged at 4800 rpm for 15 min at 25°C to obtain a supernatant, and then the supernatant was filtered using 5C filter paper (ADVANTEC quantitative filter paper) with a pore size of 110 mm to obtain a concentrated filtrate with a dry matter mass concentration of 10 mg / mL.

[0091] According to the mass percentage, 2% of the above outer membrane vesicle concentrate, 1% of the above concentrated filtrate and the balance of water are mixed and then allowed to stand for 2 hours to obtain a tea extract.

[0092] S2 Skin Care Composition Preparation

[0093] According to the weight ratio, 0.5 parts of the above-mentioned tea extract, 2 parts of polysaccharide-modified liposomes, 0.2 parts of Ganoderma lucidum extract, 1 part of glycerin (stabilizer), 5 parts of caprylic / capric / lauric triglyceride (stabilizer) and 10 parts of 1,3-butylene glycol (preservative) were mixed, and then ultrasonically treated at room temperature for 2 minutes and homogenized at 2000 rpm for 3 minutes to obtain a skin care composition.

[0094] The preparation steps of the polysaccharide modified liposomes include: dissolving lecithin in anhydrous ethanol to obtain a mixed solvent; then removing the anhydrous ethanol in the mixed solvent by a rotary evaporator until a transparent lipid film is formed; then adding a sterile phosphate buffer solution (PBS) with a pH of 7.0 to the lipid film and rotating and hydrating it in a 50°C water bath at normal pressure, so that the lipid film is fully dissolved in the PBS solution to obtain a multilamellar liposome; then ultrasonically treating the multilamellar liposome in an ice water bath for 2 minutes and then placing it in a 50°C water bath for 2 hours to obtain a liposome. Weigh water-soluble chitosan (in terms of mass percentage, the mass ratio of water-soluble chitosan to liposome is 0.001:1) and dissolve it in sterile water, stirring to obtain a chitosan solution; stirring and mixing the chitosan solution and the above liposome solution for 0.5 hours to obtain a polysaccharide modified liposome.

[0095] Test example

[0096] 1. Performance test of tea leaf extract

[0097] Ability of tea extract to inhibit iNOS expression

[0098] Test principle: Nitric oxide (NO) plays an important regulatory role in skin inflammation, regulating inflammation by affecting the cyclooxygenase / prostaglandin pathway, inflammatory cell migration and cytokine production. NO is mainly synthesized by nitric oxide synthase (NOS) in the body, including cytokine-inducible nitric oxide synthase (iNOS). Studies have found that the expression of iNOS is significantly increased under the action of stimulating factors such as lipopolysaccharide (LPS). The reason is that after LPS binds to the TLR4 receptor on the cell surface, it causes IKK activation, which ultimately leads to NF-κB entering the nucleus and binding to the iNOS promoter, promoting iNOS gene transcription. Therefore, by monitoring the changes in NO content and the expression of iNOS mRNA in LPS-induced mouse macrophages RAW264.7, the ability of tea extract to inhibit iNOS can be tested, thereby verifying its anti-inflammatory effect.

[0099] Test method:

[0100] Cell culture and cell survival rate detection: RAW264.7 mouse macrophages were revived with DMEM medium containing 1% double antibody and cultured in a 37°C, 5% CO2 incubator for 1 to 2 days. When grown to a density that can be subcultured, they were inoculated into a 24-well plate on average, 500 μL per well, and placed in an incubator for culture for 24 hours. The cell culture medium was discarded, and the corresponding drug solution was added to obtain a blank group, a stimulation group, and an experimental group, respectively, wherein the corresponding drug solution of the blank group was the culture medium, the corresponding drug solution of the stimulation group was 50 ng / mL LPS, and the corresponding drug solution of the experimental group was the tea extract in Example 1 + 50 ng / mL LPS (wherein the mass proportion of the tea extract in the drug solution was 0.5%). Continue to culture for 24 hours, add 100 μL of 5 mg / mL MTT solution to each well, and culture and incubate in the incubator for 2 hours. The supernatant was discarded, 500 μL of isopropanol was added to each well, and the cells were shaken for 30 min in the dark. The absorbance was measured at 570 nm using an enzyme reader, and the cell survival rate was calculated based on the test results (the cell survival rate of the stimulation group was used as the benchmark) and was statistically reported in Table 1.

[0101] Table 1

[0102]

[0103] As shown in Table 1, the MTT results show that the cell survival rates of the experimental group and the experimental group are basically the same, proving that the mass proportion of tea extract in the drug solution is 0.5%, which is a safe concentration, that is, the test results are reliable.

[0104] NO content determination: According to the instructions of the Griess Reagent System-16 kit, 50 μL of the cell culture medium of each sample was mixed with 50 μL of Sulfanilamide solution reagent and 50 μL of NED solution reagent in a 96-well plate according to the steps, and the standard curve was prepared according to the instructions. The absorbance value was measured at 540 nm with an ELISA reader, and then the NO content was calculated according to the test results and statistically reported in Table 2.

[0105] Table 2

[0106]

[0107] As shown in Table 2, compared with the blank group, the NO content of the cells in the stimulation group increased to 18.63 μmol / L after 24 hours of LPS treatment, and the NO production increased significantly, indicating that the stimulation model is effective. Compared with the stimulation group, the NO content of the experimental group decreased significantly, proving that the tea extract can effectively inhibit the increase of NO content induced by LPS.

[0108] Detection of iNOS mRNA expression by timed quantitative PCR: Total RNA was extracted according to the instructions of TRIZOL extraction reagent. After a certain proportion of dilution, the concentration of iNOS mRNA extracted from each sample was measured so that the 260 / 280 value was between 1.8 and 2.0. The primer sequences used are shown in Table 3. After measuring the concentration, each sample was diluted with RNase-free water according to the lowest concentration level. Take 10 μL of each diluted sample for gel electrophoresis, and the rest was stored in a -80°C freezer. The PCR reaction system conditions are as follows: pre-denaturation at 94°C for 30s, denaturation at 94°C for 30s, annealing at 60°C for 30s, extension at 72°C for 1min, 25 cycles, and finally extension at 72°C for 7min. After the reaction, the reaction product was identified by 1% agarose gel electrophoresis (95V, 30min), and grayscale scanning analysis was performed using a gel imager. According to the analysis results, the expression of iNOS mRNA is summarized in Table 4.

[0109] Table 3

[0110]

[0111] Table 4

[0112]

[0113] As shown in Table 4, compared with the blank group, the iNOS mRNA expression in the stimulation group increased significantly after 24 hours of LPS treatment, indicating that the stimulation model is effective. Compared with the stimulation group, the iNOS mRNA expression in the experimental group decreased significantly, proving that the tea extract can effectively inhibit the expression of iNOS, thereby having an anti-inflammatory effect.

[0114] The ability of B. truncatum tea extract to inhibit TRPV1 expression

[0115] Test principle: Studies have shown that TRPV1 can be widely expressed in fibroblasts and keratinocytes, and has the function of regulating the normal physiological functions of the skin. Another study found that the mouse model of atopic dermatitis was induced by treating with TRPV1 antagonist ruthenium red (RR), and it was found that the scratching behavior of the mice was significantly less than that of the control group. This suggests that inhibiting TRPV1 expression may reduce skin sensitivity and itching. By detecting the expression of TRPV1 mRNA, the inhibitory effect of tea extract on TRPV1 can be tested, thereby verifying its soothing effect.

[0116] Test method:

[0117] Cell culture and cell viability detection: Please refer to Experimental Example 1 for details.

[0118] TRPV1 mRNA expression was detected by timed quantitative PCR: Total RNA was extracted according to the instructions of TRIZOL extraction reagent. After a certain proportion of dilution, the concentration of TRPV1 mRNA of each sample extracted was measured so that the 260 / 280 value was between 2.0 and 2.2. The primer sequences used are shown in Table 5. After measuring the concentration, each sample was diluted with RNase-free water according to the lowest concentration level. Take 10 μL of each diluted sample for gel electrophoresis, and the rest was frozen at -80°C. The PCR reaction system conditions are as follows: pre-denaturation at 95°C for 2min, denaturation at 95°C for 15s, annealing at 60°C for 30s, extension at 60°C for 30s, and 40 cycles. After the reaction, the reaction product was identified by 1% agarose gel electrophoresis (95V, 30min), grayscale scanning analysis was performed using a gel imager, and the TRPV1 mRNA expression was summarized in Table 6 according to the analysis results.

[0119] Table 5

[0120]

[0121] Table 6

[0122]

[0123] As shown in Table 6, compared with the blank group, the expression of TRPV1 mRNA in the stimulation group increased significantly after 24 hours of LPS treatment, indicating that the stimulation model is effective. Compared with the stimulation group, the expression of TRPV1 mRNA in the experimental group decreased significantly, proving that the tea extract can effectively inhibit the expression of TRPV1, thereby having the effect of improving skin sensitivity and itching (i.e., soothing allergies).

[0124] From the above content, it can be seen that tea extract can synchronously and effectively inhibit the expression of iNOS and TRPV1, so that the skin care extract products have anti-inflammatory, soothing and anti-wrinkle effects.

[0125] 2. Performance test of skin care product composition

[0126] The skin care composition prepared in Example 1 was used as an additive, and then a skin care emulsion was prepared according to the formula in Table 7 and a conventional preparation process, and then various properties of the skin care emulsion were tested.

[0127] Table 7

[0128]

[0129]

[0130] A Skin soothing and repairing performance test:

[0131] Experimental principle: Studies have found that long-term exposure of the skin to ultraviolet rays will cause the skin to synthesize prostaglandins and active transmitters, dilate the skin capillaries, and increase permeability, thus causing erythema reactions accompanied by symptoms such as itching and pain. This is a self-degrading inflammatory response of the skin to ultraviolet damage. By irradiating the skin with ultraviolet rays to damage the skin epidermis and cause erythema reactions, and then applying the prepared skin care lotion, its efficacy in soothing and repairing the skin can be verified.

[0132] Determine the erythema value and take photos for recording: 30 subjects with no obvious marks on their arms were selected, with an age range of 20-45 years old, male / female. The inner side of the forearm was used as the test area, and the area to be irradiated was enclosed on the forearm with low-allergenic tape, and the rest was superimposed on it with black tape to play a sunscreen role. Place the UVB lamp above the forearm and irradiate it evenly and parallelly until visible erythema appears. Measure and record the erythema value before irradiation, recorded as the blank value; measure and record the erythema value after irradiation, recorded as the basic value; measure and record the erythema value again 5 hours after applying the sample, recorded as the final value, and compare the changes in the erythema value. Among them, the blank group was not applied with any sample, the experimental group contained the skin care composition in the emulsion, and the control group did not contain the skin care composition in the emulsion, and the test results were statistically summarized in Table 8.

[0133] Table 8

[0134]

[0135] See also Figure 2 (From left to right, the pictures of the three groups of samples before, after and 5 hours after UV irradiation) and Table 8, according to the changes in erythema values, all three groups of samples developed erythema after UVB irradiation, and there was no significant difference in the erythema values ​​(i.e., basal values) after irradiation. 5 hours after applying the sample, the final erythema value of the blank group increased; compared with the blank group, the final erythema value of the control group decreased significantly; compared with the control group, the final erythema value of the experimental group decreased significantly, proving that the skin care composition provided by the present application can promote epidermal repair, reduce inflammatory redness symptoms, and has a skin soothing and repairing effect.

[0136] B Skin anti-wrinkle and firming performance test:

[0137] Experimental principle: Collagen fibers are the main component of the dermis of the skin. The degeneration of collagen fibers can cause wrinkles on the skin, and the matrix metalloproteins secreted by epidermal cells are involved in the degradation of collagen fibers, leading to accelerated aging of the skin. According to research results, there is a correlation between TRPV1 and matrix metalloproteins. Blocking TRPV1 or knocking out its gene can reduce the expression of matrix metalloproteins, and vice versa, it can promote the expression of matrix metalloproteins. Therefore, the skin anti-wrinkle and firming effect of the skin care composition prepared in Example 1 can be tested by this principle.

[0138] Determination of wrinkles at the corners of the eyes: Referring to the "T / CAB 0152-2022 Test Methods for Seven Efficacy of Cosmetics for Anti-wrinkle, Firming, Moisturizing, Oil Control, Repair, Nourishing, and Soothing", 30 normal and healthy subjects who met the conditions were screened, with an age range of 20 to 45 years old, male / female. The left and right corners of the eyes were selected as the test area. The samples were used once in the morning and evening every day, and the amount used each time was about the size of a grain of rice. Among them, the emulsion containing the skin care composition was used as the experimental group, and the emulsion not containing the skin care composition was used as the control group. The entire test period was 4 weeks. The wrinkle depth was measured before application and recorded as the basic value, and then measured every two weeks, and the test results were statistically reported in Table 9.

[0139] Table 9

[0140]

[0141] See also Figure 3 , Figure 4(From left to right, they are pictures of the corresponding samples at 0 days, 14 days after applying the samples, and 28 days after applying the samples, wherein each group of samples is accompanied by two pictures at the same time, the upper picture is a wrinkle plane map, used to determine the range of wrinkles; the lower picture is a wrinkle scan map, used to determine the depth of wrinkles) and Table 9, from the changes in wrinkle depth, it can be seen that 28 days after applying the samples, the transverse wrinkle depth in the test area of ​​the control group and the experimental group is improved, and the wrinkle improvement effect of the experimental group is more significant. There is a significant difference in the wrinkle depth values ​​between the control group and the experimental group, which can prove that the skin care composition provided by the present application can inhibit and improve the degradation of collagen, and exert the anti-wrinkle and firming effect of the skin.

[0142] C Skin moisturizing performance test

[0143] Experimental principle: The skin contains a structure that retains and releases moisture. In the stratum corneum, the moisture content gradually decreases from the bottom layer to the upper layer. The bottom layer of the stratum corneum acts as a water barrier, and water penetrates from the inside to the outer layer and the surrounding atmosphere. The water content of the stratum corneum of the skin is an important parameter for maintaining the moisture balance of the skin. When the water content of the stratum corneum of the skin is low, the skin will become dry, and phenomena such as desquamation and wrinkles will occur, which will increase the risk of skin infection in the long run. Therefore, based on the capacitance method to measure the moisture content of the stratum corneum of the skin, the skin moisturizing ability of the skin care composition prepared in Example 1 can be tested.

[0144] Determination of stratum corneum water content: Referring to "T / CAB 0152-2022 Test Methods for Seven Efficacy of Cosmetics for Anti-wrinkle, Firming, Moisturizing, Oil Control, Repair, Nourishing, and Soothing", 30 normal and healthy subjects who met the conditions were screened, with an age range of 20 to 45 years old, male / female. The left and right cheeks were selected as the test area. The samples were used once in the morning and evening every day, and the amount used each time was about the size of a grain of rice. Among them, the emulsion containing the skin care composition was used as the experimental group, and the emulsion not containing the skin care composition was used as the control group. The entire test period was 4 weeks. The moisture content was measured before application and recorded as the basic value, and then measured every two weeks, and the test results were statistically reported in Table 10.

[0145] Table 10

[0146]

[0147]

[0148] Referring to Table 10, it can be seen from the changes in the water content of the stratum corneum of the skin that 28 days after applying the sample, the water content of the stratum corneum of the skin of the experimental group increased by 13.5, which was significantly higher than that of the control group. There was a significant difference in the water content values ​​between the control group and the experimental group, which can prove that the skin care composition provided in the present application can increase the water content of the stratum corneum and play a role in long-term moisturizing of the skin.

[0149] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

Claims

1. A use of tea extract in the preparation of a skin care composition having the efficacy of inhibiting the expression of cytokine-inducible nitric oxide synthase and capsaicin receptor.

2. The use according to claim 1, characterized in that: The preparation steps of the tea extract include: Extracting tea leaves powder by water extraction to obtain extract; filtering the extract to obtain outer membrane vesicles and a filtrate respectively; The outer membrane vesicles at the filter element are rinsed with water and collected to obtain an outer membrane vesicle concentrate; The outer membrane vesicle concentrate, the filtrate and water are mixed and then allowed to stand to obtain the tea extract.

3. The use according to claim 2, characterized in that: The step of filtering the extract comprises: The extract is filtered using 5C filter paper to obtain a primary filtrate; Performing secondary filtration on the primary filtrate using a microfiltration membrane to obtain a secondary filtrate; Using an ultrafiltration membrane to perform three-stage filtration on the secondary filtrate to obtain the outer membrane vesicles and the filtrate respectively; Optionally, in the three-stage filtration step, the ultrafiltration membrane used is an external pressure ultrafiltration membrane; Optionally, in the three-stage filtration step, the filtration pressure is 0.1-0.6 MPa.

4. The use according to claim 3, characterized in that: The volume ratio of the outer membrane vesicle concentrated solution to the secondary filtrate is 1:(0.2-0.5).

5. The use according to claim 4, characterized in that: Before the step of mixing the outer membrane vesicle concentrate, the filtrate and water and then placing them aside for a static treatment, the method further includes a step of concentrating the filtrate so that the mass concentration of dry matter in the filtrate is 8 to 12 mg / mL.

6. The use according to claim 5, characterized in that: The step of mixing the outer membrane vesicle concentrate, the filtrate and water and then placing them in a static state comprises: According to mass percentage, 1-5% of the outer membrane vesicle concentrate, 0.5-2% of the filtrate and the balance of the water are mixed and then allowed to stand.

7. The use according to any one of claims 2 to 6, characterized in that: In the step of standing treatment, the standing time is 2 to 4 hours.

8. The use according to any one of claims 1 to 6, characterized in that: The skin care composition comprises, by weight, 0.5 to 8 parts of the tea extract, 2 to 10 parts of polysaccharide-modified liposomes, 0.1 to 1 part of Ganoderma lucidum extract, 5 to 10 parts of a stabilizer and 5 to 10 parts of a preservative.

9. The use according to claim 8, characterized in that: The skin care product composition is used for preparing skin care products.

10. The use according to claim 9, characterized in that: The skin care product composition accounts for 0.5-1% by weight in the skin care product.

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