CN117815093A

CN117815093A - Expression promoter of proline-rich small protein gene SPRR2A

Info

Publication number: CN117815093A
Application number: CN202311867827.8A
Authority: CN
Inventors: 殷庆飞; 陈园园
Original assignee: Shanghai Jahwa United Co Ltd
Current assignee: Shanghai Jahwa United Co Ltd
Priority date: 2023-12-29
Filing date: 2023-12-29
Publication date: 2024-04-05

Abstract

The invention provides an expression promoter of a small protein gene SPRR2A with rich proline in keratinocytes, wherein the expression promoter is amentoflavone. Preferably, amentoflavone is used at a concentration of at least 0.000156mg/ml. The invention also relates to application of amentoflavone in preparing skin external preparation with the effect of promoting expression of small protein gene SPRR2A rich in proline in keratinocyte. Preferably, the amentoflavone is contained in the skin external preparation in an amount of 0.00001 to 10 wt%.

Description

Expression promoter of proline-rich small protein gene SPRR2A

Technical Field

The invention relates to the field of research on efficacy of raw materials of cosmetics, in particular to a natural compound amentoflavone which is derived from plants and is used for up-regulating expression of a proline-rich small protein SPRR2A gene of skin epidermis keratinocytes, and the amentoflavone can be used as an accelerator for expression of the proline-rich small protein SPRR2A gene of skin epidermis keratinocytes.

Background

Human skin is divided into three parts of epidermis, dermis and subcutaneous tissue, wherein the epidermis is at the outermost side of human body, has the function of preventing the loss of nutrients and moisture in skin, and has the function of defending and protecting the external environment such as physics, chemistry and microorganism. The epidermis is divided into basal layer, ratchet layer, granular layer and cuticle layer from inside to outside, wherein the cuticle layer has a structure similar to a brick wall structure and is composed of keratinocytes and intercellular lipids, and can protect the inner skin tissues such as the inner side of the epidermis, dermis and the like.

Epidermal differentiation is a highly morphologically changing process, in which keratinocytes gradually differentiate from basal to stratum corneum, and in which specific proteins are co-involved. Among them, keratinocyte envelope formation is an important process in keratinocyte differentiation, and is a process of forming a mechanical protective structure of the epidermis stratum corneum.

The epidermis is mainly composed of keratinocytes, accounting for about 90% of the number of epidermal cells, and the differentiation of the epidermis is mainly related to the differentiation of keratinocytes. Keratinocyte differentiation is a process involving hundreds of genes that are involved in and regulated. This process includes lamellar platelet formation, formation of keratinized envelope, lipid synthesis, and the like. For example, proline-rich small proteins (SPRPs), a class of keratinocyte structural proteins, are associated with the formation of a keratinized envelope, which can be chemically cross-linked with membrane proteins by transglutaminase to form an insoluble keratinized envelope under the plasma membrane. The proline-rich small protein SPRR2A is encoded by the SPRR2A gene, whose expression is closely related to terminal differentiation of keratinocytes in vivo and in vitro.

The main components of the keratinized envelope also include the protein papilionamine (LOR), which is expressed mainly in the stratum granulosum and stratum corneum of the epidermis of the skin, accounting for approximately 80% of its protein content. The pappalin LOR consists of repeated glycine loops (glycine-serine), and the pappalin and proline-rich small proteins (SPRPs) are crosslinked with cell scaffolds outside the envelope under the catalysis of transglutaminase (TGM 1 and TGM 3) to play a role in mechanical reinforcement in the cytoplasmic surface of the cuticle.

Because the proteins play an important role in the skin epidermis differentiation process and epidermis structure and function, active substances/components capable of regulating and controlling the expression of genes related to the epidermis cell differentiation are searched, researched and developed, the expression of the proteins in the skin epidermis is improved, and the proteins have positive significance and important application value for promoting the reconstruction of the damaged skin epidermis function and recovering the normal physiological function of the skin epidermis.

Amentoflavone is one of the main active ingredients in Selaginella (Selaginella) plants, and has antioxidant, antiinflammatory, antibacterial, and anticancer effects. Amentoflavone is a natural biflavanoid compound which was originally isolated from tamariskoid spikemoss, red branch tamariskoid spikemoss and ginkgo leaf, and thereafter, amentoflavone was also sequentially extracted from 120 or more plants such as cypress, wood bamboo, induced grass, hypericum perforatum, etc. However, there are few studies and patents on amentoflavone in promoting epidermal cell differentiation.

In the process of researching the function and activity of amentoflavone in epidermis, it is unexpectedly found that amentoflavone has the effect of increasing the expression of a differentiation related gene, namely, a proline-rich small protein gene SPRR2A, in a cytoplasmic cell, and has the effects of promoting the differentiation of epidermal keratinocytes and improving the function of epidermis.

Disclosure of Invention

In one aspect, the invention provides an expression promoter of a proline-rich small protein gene SPRR2A in keratinocytes, wherein the expression promoter is amentoflavone.

In a preferred embodiment, the expression enhancer is used at a concentration of at least 0.000156mg/mL, preferably 0.000156-0.01mg/mL.

In a preferred embodiment, the expression enhancer is used at a concentration of 0.000625-0.01mg/mL.

In a preferred embodiment, amentoflavone purity is greater than or equal to 98wt%.

In a preferred embodiment, the relative expression level of the amentoflavone-promoting keratinocyte-rich proline miniprotein gene SPRR2A is greater than or equal to 116%, preferably 116-202%.

In another aspect, the invention also relates to the use of amentoflavone in the preparation of a skin external preparation having the effect of promoting expression of the proline-rich small protein gene SPRR2A in keratinocytes.

In a preferred embodiment, amentoflavone is present in the skin external preparation in an amount of 0.00001 to 10% by weight, preferably 0.002 to 1% by weight.

In a preferred embodiment, the skin external agent is selected from the group consisting of: face cream, milky lotion, jelly, pack, face toilet, pack, aerosol cleansing foam, spray, body wash, or facial cleanser.

Drawings

FIG. 1 shows a sample of amentoflavone.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. For the purposes of the present invention, the following terms are defined below.

The term "about" as used herein refers to an amount, level, value, dimension, size, or use that may differ by up to 30%, 20%, or 10% from the amount, level, value, dimension, size, or use of a reference. The percentages used herein are by weight unless otherwise indicated.

Throughout the specification and claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The invention is based on the following unexpected findings: the amentoflavone has the effect of increasing expression of differentiation related genes, namely proline-rich small protein genes SPRR2A, in the cytoplasmic forming cells, and has the effects of promoting the differentiation of epidermal keratinocytes and improving the epidermal functions.

The invention has the beneficial effects that the amentoflavone which is a natural compound derived from traditional Chinese medicines is found, can be used as an accelerator for expression of a differentiation gene-rich proline small protein gene SPRR2A in keratinocytes, has the effects of accelerating the differentiation of epidermal keratinocytes and improving the epidermal functions, has important application value, can be applied to development and application of various cosmetic products, and has the effects and effects of reconstructing the epidermal functions of the skin and recovering the normal physiological functions of the skin.

Amentoflavone from amentoflavone

The present invention surprisingly found that amentoflavone has the effect of upregulating expression of the proline-rich small protein gene SPRR2A, a gene associated with differentiation in cytoplasmic cells. Therefore, amentoflavone can be used as an accelerator for expression of a differentiation gene-rich proline small protein gene SPRR2A in keratinocytes, applied to development and application of various cosmetic products, and endowed with the effects and functions of reconstructing the functions of the epidermis of the skin and recovering the normal physiological functions of the epidermis of the skin.

The application discovers for the first time that amentoflavone has the effects of promoting the differentiation of epidermal keratinocytes and improving the epidermal functions, and has important application value. In a preferred embodiment, amentoflavone is derived from Selaginella tamariscina.

In some embodiments, amentoflavone has the effect of promoting expression of the proline-rich small protein gene SPRR 2A. In some embodiments, amentoflavone is used at a concentration of at least 0.000156mg/ml, preferably at least 0.000625mg/ml.

In some embodiments, amentoflavone is used at a concentration of 0.000156-0.01mg/ml, preferably 0.000625-0.01mg/ml.

In some embodiments, amentoflavone is preferably used at a concentration of less than 0.01mg/ml.

External preparation for skin

In some embodiments, amentoflavone may be used in the preparation of external skin preparations. The skin external preparation is preferably a cosmetic composition including, but not limited to, products in the form of face cream, milky lotion, jelly, lotion, essence, pack, eye cream, aerosol (cleansing foam), spray, body wash, facial cleanser, and the like.

The weight percentage of amentoflavone in the skin external agent is 0.00001% -10% (w/w), preferably 0.0001% -5% (w/w), more preferably 0.001% -2% (w/w), and most preferably 0.002% -1% (w/w).

In particular embodiments, amentoflavone may be used in the preparation of personal care cleansing products. The personal cleansing care product is preferably a rinse-off cleansing product composition including, but not limited to, the preparation of products in the form of baby body washes, shampoos, shampoo body washes, child and adult body washes, shampoos, hand washes, and facial cleansers. The plant extracts of the present application are also intended to be included in the context of the modification of the product morphology, for example in the form of powders, blocks, pastes, bars, flakes, etc., or intermediates.

The amentoflavone is present in the personal care composition in an amount of from 0.0001% to 10% (w/w), preferably from 0.0005% to 5% (w/w), more preferably from 0.001% to 2% (w/w), and most preferably from 0.002% to 1% (w/w).

The external preparation for skin is a general term for all components usually used outside the skin, and may be, for example, a cosmetic composition. The cosmetic composition may be basic cosmetic, facial makeup cosmetic, body cosmetic, hair care cosmetic, etc., and its dosage form is not particularly limited and may be reasonably selected according to different purposes. The cosmetic composition also contains various cosmetically acceptable medium or matrix excipients depending on dosage form and purpose.

The external preparation for skin comprising amentoflavone can be topically applied to human skin and/or hair. The skin external preparation may further comprise a cosmetically acceptable topical carrier, which may be about 50% to about 99.99% by weight of the skin external preparation (e.g., about 80% to about 99% by weight of the skin external preparation). In a preferred embodiment of the invention, the cosmetically acceptable topical carrier comprises water. The cosmetically acceptable topical carrier may include one or more materials selected from the group consisting of moisturizers, emollients, oils, humectants, and the like. In one embodiment, the cosmetically acceptable topical carrier includes a substrate such as a nonwoven or film material.

Skin external preparations may be formulated into a variety of product types including, but not limited to, lotions, creams, gels, sticks, sprays, ointments, cleansing liquid lotions and solid soaps, shampoos and hair conditioners, hair fixatives, pastes, foams, powders, mousses, shave creams, wipes, patches, hydrogels, film-forming products, masks and skin films, films and cosmetics such as foundations and mascaras. These product types may contain several types of cosmetically acceptable topical carriers including, but not limited to, solutions, suspensions, emulsions (e.g., microemulsions and nanoemulsions), gels, solids, and liposomes.

The skin external preparation containing amentoflavone can be formulated into solution. The solution typically comprises an aqueous or organic solvent (e.g., about 50% to about 99.99% or about 90% to about 99% of a cosmetically acceptable aqueous or organic solvent). Examples of suitable organic solvents include propylene glycol, polyethylene glycol, polypropylene glycol, glycerol, 1,2, 4-butanetriol, sorbitol esters, 1,2, 6-hexanetriol, ethanol and mixtures thereof.

The skin external preparation may be formulated as a solution containing an emollient. Such skin external preparations preferably comprise from about 2% to about 50% of one or more emollients. As used herein, "emollient" refers to a substance used to prevent or reduce dryness, for example, by preventing the loss of skin moisture through the skin. Examples of emollients include, but are not limited to, vegetable oils, mineral oils, aliphatic esters, and the like.

Lotions can be prepared from such solutions. Lotions typically contain from about 1% to about 20% (e.g., from about 5% to about 10%) of one or more emollients and from about 50% to about 90% (e.g., from about 60% to about 80%) of moisture.

Another type of product that can be formulated from solutions is a cream. A cream typically contains from about 5% to about 50% (e.g., from about 10% to about 20%) of one or more emollients and from about 45% to about 85% (e.g., from about 50% to about 75%) of moisture.

While it is preferred that the skin external preparation comprising amentoflavone comprises water, the skin external preparation may alternatively be anhydrous or ointments that do not comprise water but instead are organic and/or silicone solvents, oils, fats and waxes. Ointments may contain simple bases of animal or vegetable oils or semi-solid hydrocarbons. Ointments may contain from about 2% to about 10% of one or more emollients and from about 0.1% to about 2% of one or more thickeners.

The skin external preparation can be formulated as an emulsion. If the topical carrier is an emulsion, from about 1% to about 10% (e.g., from about 2% to about 5%) of the topical carrier contains one or more emulsifying agents. The emulsifier may be nonionic, anionic or cationic. Examples of suitable emulsifiers include those commonly identified as suitable emulsifiers in the personal care and cosmetic formulations arts.

Lotions and creams can be formulated as emulsions. Typically such lotions contain from 0.5% to about 5% of one or more emulsifying agents. Such creams typically contain from about 1% to about 20% (e.g., from about 5% to about 10%) of one or more emollients; about 20% to about 80% (e.g., 30% to about 70%) water; and from about 1% to about 10% (e.g., from about 2% to about 5%) of one or more emulsifiers.

Oil-in-water and water-in-oil single emulsion skin care formulations, such as lotions and creams, are well known in the cosmetic arts and can be used in the present invention. Multiple emulsion skin external preparations (e.g., water-in-oil-in-water and oil-in-water) are also useful in the present invention. Typically, such single-phase or multiple-phase emulsions contain moisture, emollients, and emulsifiers as their essential ingredients.

The skin external preparation containing amentoflavone may also be formulated as a gel (e.g., an aqueous gel, an alcoholic gel, an alcohol/water gel, or an oily gel using a suitable gelling agent). Suitable gelling agents for aqueous and/or alcoholic gels include, but are not limited to, natural gums, acrylic acid and acrylate polymers and copolymers, and cellulose derivatives (e.g., hydroxymethyl cellulose and hydroxypropyl cellulose). Suitable gellants for oils (e.g., mineral oils) include, but are not limited to, hydrogenated butene/ethylene/styrene copolymers and hydrogenated ethylene/propylene/styrene copolymers. Such gels typically contain between about 0.1% and 5% by weight of such gelling agents.

The external preparation for skin containing amentoflavone may also be formulated as a solid preparation (e.g., a wax-based stick, bar soap, powder, or wipe containing powder).

In addition to the above components, the skin external preparations usable in the present invention may contain various other oil-soluble substances and/or water-soluble substances which are conventionally used in skin external preparations for use on the skin and hair at levels determined in the technical field thereof.

The skin external agent of the present invention may contain additional components commonly found in skin care compositions, such as emollients, skin conditioning agents, emulsifiers, preservatives, antioxidants, fragrances, chelating agents, and the like, as long as they are physically and chemically compatible with the other components in the skin external agent and do not affect the effects of amentoflavone of the present invention.

In some embodiments of the skin external preparation of the present invention, one or more preservatives may be used. Suitable preservatives include p-hydroxyacetophenone, alkyl C1-C4 p-hydroxybenzoates and phenoxyethanol. The preservative is used in an amount of about 0.5 to about 2 wt%, preferably about 0.5 to 1 wt%, based on the total weight of the composition.

In one example of the skin external agent of the present invention, one or more antioxidants may be used. Suitable antioxidants include Butylated Hydroxytoluene (BHT), ascorbyl palmitate (BHA), butylated hydroxyanisole, phenyl-alpha-naphthylamine, hydroquinone, propyl gallate, nordihydroguaiaretic acid, vitamin E or derivatives of vitamin E, vitamin C and its derivatives, calcium pantothenate, green tea extracts and mixed polyphenols, and mixtures of the foregoing. The antioxidants are used in an amount ranging from about 0.02 to 0.5 weight percent, more preferably from about 0.002 to 0.1 weight percent, based on the total weight of the composition.

In one example of the skin external agent of the present invention, one or more emollients may be used which act as lubricants to reduce flaking and improve the appearance of the skin by their ability to remain on the skin surface or in the stratum corneum. Typical emollients include fatty esters, fatty alcohols, mineral oils, polyether siloxane copolymers, and the like. Examples of suitable emollients include, without limitation, polypropylene glycol ("PPG") -15 stearyl ether, PPG-10 cetyl ether, steareth-10, oleth-8, PPG-4 lauryl ether, vitamin E acetate, lanolin, cetyl alcohol, cetostearyl alcohol ethyl hexanoate, cetostearyl alcohol, glyceryl stearate, octyl hydroxystearate, dimethylpolysiloxane, and combinations thereof. Cetyl alcohol, cetostearyl alcohol ethyl hexanoate, cetostearyl alcohol, glycerol stearate, and combinations thereof are preferred. When used, the emollient is in an amount ranging from about 0.1 to about 30 weight percent, preferably from about 1 to about 30 weight percent, based on the total weight of the composition.

In one example of the skin external agent of the present invention, one or more moisturizers may be used. Humectants, also known as humectants, help to enhance the effectiveness of emollients, reduce flaking, stimulate removal of constituent scales and enhance skin feel. Polyols may be used as humectants including, but not limited to, glycerin, polyalkylene glycols, alkylene polyols and derivatives thereof, including butylene glycol, propylene glycol, dipropylene glycol, polyglycerol, polyethylene glycol and derivatives thereof, sorbitol, hydroxypropyl sorbitol, hexylene glycol, 1, 3-dibutylene glycol, 1,2, 6-hexanetriol, ethoxylated glycerin, propoxylated glycerin and combinations thereof. When used, the humectant is present in an amount of about 0.1 to about 20 weight percent, preferably about 1 to about 15 weight percent, based on the total weight of the composition.

In one example of the skin external agent of the present invention, one or more emulsifying agents may be used. The emulsifier may be used in an effective stabilizing amount. Preferably, the emulsifier is used in an amount of about 1.0 to about 10.0 wt%, more preferably about 3.0 to about 6.0 wt%, based on the total weight of the composition. Any emulsifier that is compatible with the components of the composition may be used. Suitable emulsifiers include stearic acid, cetyl alcohol, glyceryl stearate, lecithin, stearyl alcohol, steareth-2, steareth-20, acrylic/C10-30 alkanol acrylate cross-linked polymers, and combinations thereof.

In one example of the skin external agent of the present invention, one or more pH adjusting agents may be used. The pH adjuster useful in the skin external preparation of the present invention includes tromethamine. When used, the pH adjustor is used in an amount of about 0.1 to about 2 weight percent, preferably about 0.1 to about 1 weight percent, based on the total weight of the composition.

In one embodiment of the present invention, the skin external preparation comprises acrylic/C10-30 alkanol acrylate cross-linked polymer, glycerol, p-hydroxyacetophenone, glycerol stearate and lecithin, cetyl/stearyl alcohol, cetostearyl alcohol ethyl hexanoate, tromethamine or combinations thereof.

Detailed Description

The present invention will be further illustrated with reference to specific examples and comparative examples. However, these examples and comparative examples are only for illustrating the present invention, and do not limit the scope of the present invention. The experimental methods in the following examples and comparative examples, in which specific conditions are not specified, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. All percentages and parts are by weight unless otherwise indicated.

Example 1: cytotoxicity test

The invention adopts an MTT method to carry out cytotoxicity test on amentoflavone, and analyzes the maximum nontoxic dose of amentoflavone in a keratinocyte culture system.

Cytotoxicity assays refer to the use of cell culture techniques to evaluate cell death, inhibition of cell growth, or other effects on cells caused by a test sample using a cytotoxicity assay. In the invention, the maximum nontoxic dose of the sample in the test cell system is calculated through cytotoxicity test analysis, so that guidance is provided for the concentration of the sample in other efficacy tests, and the feasibility of subsequent tests and the validity of data are ensured. The MTT method is adopted for cytotoxicity test in the invention.

The MTT method is a commonly used method for testing sample cytotoxicity and cell survival and proliferation. MTT is known as 3- (4, 5-Dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide, and the chemical name of Chinese is 3- (4, 5-Dimethyl thiazole-2) -2, 5-Dimethyl tetrazolium bromide, and is a yellow dye. The main principle is that the activity of enzyme in cell mitochondria is utilized to convert specific tetrazolium salt, and then the tetrazolium salt is detected by an enzyme-labeled instrument. The specific principle is that succinate dehydrogenase in the mitochondria of living cells is able to reduce exogenous MTT to water insoluble blue-violet crystalline formazan, deposited in cells, whereas dead cells do not. Dimethyl sulfoxide (DMSO) can solubilize formazan deposited in cells to form a purple solution, and then detect OD values at 570nm wavelength (490 nm also can be used) in an enzyme-linked immunosorbent assay, which can indirectly reflect the number of living cells. The amount of MTT crystals formed is proportional to the number of cells over a range of cell numbers.

1. Cells, instruments and reagents

And (3) cells: keratinocytes were derived from Guangdong Boxi Biotechnology Co.

The main instrument is as follows: CO ₂ Incubator (150I) was purchased from Thermo corporation, U.S., ultra clean bench (SW-CJ-1F) was purchased from Atai air technologies, inc., suzhou, sujing group, and microplate reader (Epoch) was purchased from BioTek corporation, U.S., inverted microscope (CKX 53) Japan Olympus corporation.

The main reagent comprises: amentoflavone standard (CAS number 1617-53-4, purity 98%) was purchased from Nanjing Source plant Biotechnology Co., ltd., kcGrow broth (cat number 08030021) was purchased from Guangdong Boxi Biotechnology Co., ltd., MTT (cat number M5655) was purchased from Sigma Co., USA, DMSO (cat number D4540-1L) was purchased from Sigma Co., USA, and PBS (cat number P1010) was purchased from Beijing Soy Bao technology Co., ltd.).

2. Test method

1) Cell inoculation: after resuscitating the cells, inoculating the cells to a 96-well plate when the plating rate reaches about 60%, and CO ₂ Incubator (37 ℃, 5% CO) ₂ ) Incubate overnight.

2) Test grouping: the test set-up was zeroed, solvent control and sample. In the sample group, 8 concentration gradients were set for each sample, and 3 duplicate wells were set for each concentration gradient.

3) Preparing liquid: sample working solutions of different concentrations were prepared according to the test concentration profile (table 1). If the sample is insoluble in water, the mother solution with the corresponding concentration of 100 times is prepared by DMSO, and then the mother solution is diluted to the required concentration by culture medium.

Table 1: condition of amentoflavone concentration

4) Administration: and when the cell plating rate in the 96-well plate reaches 40% -60%, the administration is carried out. 200. Mu.L of culture solution was added to each well of the solvent control group; 200 mu L of culture solution containing samples with corresponding concentrations is added into each hole of the sample group; the zeroed group was inoculated without cells and only 200. Mu.L of cell culture medium was added. After completion of the dosing, the 96-well plate was placed in CO ₂ Incubator (37 ℃, 5% CO) ₂ ) Is cultured for 24 hours.

5) And (3) detection: after incubation of the cells for 24h, the supernatant was discarded, MTT working solution (0.5 mg/mL) was added, incubated at 37℃for 4h in the absence of light, after incubation, the supernatant was discarded, 150. Mu.L of DMSO was added per well, and the OD was read at 490 nm.

3 computational analysis

1) Cell relative viability calculation: according to the calculation of the formula,

2) Maximum non-toxic dose calculation for samples: according to the formula, the cell viability is 70 percent

Wherein:

AL-cell viability value (%) corresponding to the maximum sample concentration having a cell viability value higher than 70%;

AH-cell viability (%) corresponding to a minimum sample concentration having a cell viability value of less than 70%;

CH-cell viability value below 70% minimum sample concentration (mg/mL);

CL-cell viability values were higher than 70% of the maximum sample concentration (mg/mL).

4 cytotoxicity test results

Cytotoxicity test results are shown in table 2.

The calculation and analysis are carried out according to the maximum nontoxic dosage when the cell activity value is 70%, and the maximum nontoxic dosage of amentoflavone in keratinocyte treatment is 0.0127mg/mL.

Table 2: amentoflavone cytotoxicity test results

Example 2: amentoflavone-regulated SPRR2A gene test

The invention adopts a quantitative detection method of gene expression to analyze the regulation and control condition of amentoflavone on the keratinocyte SPRR2A gene.

HaCaT cells are human immortalized epidermal cells cultured by keratinocytes, are non-tumor-derived human normal skin immortalized keratinocyte cell lines, and have similar differentiation characteristics as normal human keratinocytes. HaCaT cell lines are commonly used to study the proliferation, migration and differentiation mechanisms of the skin epidermis and to study the effect of actives on keratinocytes. In the research, a culture solution containing a plurality of concentration samples is treated for 24 hours, then total ribonucleic acid (RNA) of the cells is collected, and the relative expression quantity of the proline-rich small protein gene SPRR2A is detected by adopting an RNA reverse transcription technology and a gene expression quantitative detection method, so that whether the samples have the effects of promoting the differentiation of epidermal keratinocytes and improving the epidermal functions is evaluated.

1. Cell, reagent consumable and instrument

Major cell lines: human immortalized keratinocyte cell line HaCaT was derived from the cantonese bosch biotechnology limited.

Main reagent consumable: amentoflavone samples (CAS number 1617-53-4, purity 98%) were purchased from Nanjing Source plant Biotechnology Co., ltd., fetal bovine serum, cell culture Medium high sugar DMEM, antibiotic cocktail (PSN), 0.25% pancreatin-EDTA were purchased from ThermoFisher, USA; total RNA extraction reagent TRIzol was purchased from ThermoFisher, inc., USA; reverse transcription kit iScript cDNA Synthesis Kit and fluorescenceReal-time quantitative PCR reagent iTaq ^TM UniversalGreen Supermix is available from Bio-rad, inc. of America; six well plates were purchased from Corning corporation, usa.

The main instrument is as follows: ultra-micro ultraviolet spectrophotometry NanoDrop ONE was purchased from thermo fisher company, usa, gene amplification apparatus C1000 Touch, real-time quantitative PCR apparatus CFX Connect was purchased from Bio-rad company, usa.

2. Test method

1) Cell inoculation: haCaT cells were seeded in six well plates, 3-5×10 per well ⁵ Individual cells, put into CO ₂ Incubator (37 ℃, 5% CO) ₂ ) The culture was continued for 24 hours.

2) Sample configuration: sample working solutions with different concentrations are prepared by using a cell culture medium, and the prepared concentration is smaller than the maximum nontoxic dose. If the sample is insoluble in the culture medium, the mother solution with the corresponding concentration of 100 times is prepared by DMSO, and then the mother solution is diluted to the required concentration by the culture medium.

3) Dosing and incubation: sucking old culture solution, adding 2mL culture solution into blank control hole, adding 2mL culture solution containing corresponding concentration sample into each hole of sample group, adding CO into six hole plates after administration ₂ Incubator (37 ℃, 5% CO) ₂ ) The culture was continued for 24 hours.

4) Extracting total RNA: rinsing the cells 2 times with PBS buffer, and sucking the buffer; 1mL of TRIzol reagent is added, the mixture is blown for cleavage and transferred to a 1.5mL RNase-free centrifuge tube; adding 0.2mL of chloroform into the centrifuge tube, shaking vigorously, standing for 10min, and centrifuging at a low temperature and high speed for 15min; taking 500 mu L of upper water phase by a pipette nozzle without RNase, adding equal volume of precooled isopropanol, mixing uniformly, centrifuging at low temperature and high speed for 10min, and forming white flaky precipitate on the bottom of a centrifuge tube by the centrifuged RNA; rinsing with 75% ethanol twice, removing residual ethanol with a centrifuge and a suction nozzle, uncovering, and air drying at room temperature until the solution is transparent; with 40. Mu.L of RNase-free H ₂ O was dissolved and the RNA concentration was measured by a micro-spectrophotometer.

5) Reverse transcription: 1 μg of total RNA was subjected to reverse transcription using a reverse transcription kit iScript cDNA Synthesis Kit to obtain cDNA, and the reverse transcription system is shown in Table 3.

Table 3: reverse transcription system

The reverse transcription procedure is as follows: 25 ℃ for 5min;42 ℃ for 30min;85 ℃ for 5min.

6) Quantitative detection of gene expression: real-time quantification of Polymerase Chain Reaction (PCR) reagent iTaq with fluorescence ^TM UniversalAnd (3) carrying out quantitative detection on gene expression by the Green Supermix, and analyzing and detecting the change condition of the gene expression. The fluorescent real-time quantitative polymerase chain reaction system is shown in Table 4, and the primers used for quantitative detection are shown in Table 5.

Table 4: fluorescent real-time quantitative polymerase chain reaction system

The reaction procedure was as follows: 95 ℃ for 30s;95 ℃,15s,60 ℃,30s,40 cycles; 65-95℃each time reduced by 0.5℃and (melting curve).

Table 5: primer sequence for quantitative detection of gene expression

3. Statistical analysis

The actin ACTB gene is taken as an internal reference, and the relative expression change condition of the proline-rich small protein gene SPRR2A in the sample group relative to the blank control group is analyzed by adopting a 2-delta Ct method. Analysis results are expressed as Mean ± standard deviation SD, and comparisons among groups are performed using t-test statistical analysis, with both tails, p-values <0.05 considered significant differences, and p-values <0.01 considered extremely significant differences.

4. Test results

The sample amentoflavone has up-regulation effect on the expression of the proline-rich small protein gene SPRR2A of the skin epidermis keratinocyte, and the gene test result is shown in Table 6.

The test results were as follows: compared with a blank control group, the relative expression quantity of the SPRR2A gene in the amentoflavone-0.000156 mg/mL group is 116+/-9%, the p value is less than 0.05, the difference is significant, and the amentoflavone-0.000156 mg/mL has the effect of up-regulating the expression of the SPRR2A gene; the relative expression quantity of the SPRR2A gene in the amentoflavone-0.000625 mg/mL group is 139+/-7%, the p value is less than 0.01, the difference is extremely remarkable, and the amentoflavone-0.000625 mg/mL has the effect of up-regulating the expression of the SPRR2A gene; the relative expression quantity of the SPRR2A gene in the amentoflavone-0.0025 mg/mL group is 186+/-29%, the p value is less than 0.01, the difference is extremely remarkable, and the amentoflavone-0.0025 mg/mL has the effect of up-regulating the expression of the SPRR2A gene; the relative expression quantity of the SPRR2A gene in the amentoflavone-0.01 mg/mL group is 202+/-37%, the p value is less than 0.01, the difference is extremely remarkable, and the amentoflavone-0.01 mg/mL has the effect of up-regulating the expression of the SPRR2A gene.

In conclusion, the quantitative test result of gene expression shows that amentoflavone has the effect of increasing the expression of the silk fibroin gene SPRR2A in the cytoplasmic forming cells within the range of 0.000156-0.01mg/mL, and has the effects of promoting the differentiation of epidermal keratinocytes and improving the epidermal function.

Table 6: quantitative test results of Gene expression

Comparative example 1: amentoflavone regulation LOR gene test

The invention adopts a quantitative detection method of gene expression to analyze the regulation and control condition of amentoflavone on the LOR genes of the keratinocytes.

HaCaT cells are human immortalized epidermal cells cultured by keratinocytes, are non-tumor-derived human normal skin immortalized keratinocyte cell lines, and have similar differentiation characteristics as normal human keratinocytes. HaCaT cell lines are commonly used to study the proliferation, migration and differentiation mechanisms of the skin epidermis and to study the effect of actives on keratinocytes. In the research, a culture solution containing a plurality of concentration samples is treated for 24 hours, then total ribonucleic acid (RNA) of the cells is collected, and the relative expression quantity of the papilione gene LOR is detected by adopting an RNA reverse transcription technology and a gene expression quantitative detection method, so that whether the samples have the effects of promoting the differentiation of epidermal keratinocytes and improving the epidermal functions is evaluated.

1. Cell, reagent consumable and instrument

Main reagent consumable: amentoflavone samples (CAS number 1617-53-4, purity 98%) were purchased from Nanjing Source plant Biotechnology Co., ltd., fetal bovine serum, cell culture Medium high sugar DMEM, antibiotic cocktail (PSN), 0.25% pancreatin-EDTA were purchased from ThermoFisher, USA; total RNA extraction reagent TRIzol was purchased from ThermoFisher, inc., USA; reverse transcription kit iScript cDNA Synthesis Kit and fluorescent real-time quantitative PCR reagent iTaq ^TM UniversalGreen Supermix is available from Bio-rad, inc. of America; six well plates were purchased from Corning corporation, usa.

2. Test method

5) Reverse transcription: 1. Mu.g of total RNA was subjected to reverse transcription using a reverse transcription kit iScript cDNA Synthesis Kit to obtain cDNA, and the reverse transcription system is shown in Table 7.

Table 7: reverse transcription system

6) Quantitative detection of gene expression: real-time quantification of Polymerase Chain Reaction (PCR) reagent iTaq with fluorescence ^TM UniversalGreen Supermix geneAnd (3) quantitatively detecting the expression, and analyzing and detecting the change condition of the gene expression. The fluorescent real-time quantitative polymerase chain reaction system is shown in Table 8, and the primers used for quantitative detection are shown in Table 9.

Table 8: fluorescent real-time quantitative polymerase chain reaction system

Table 9: primer sequence for quantitative detection of gene expression

3. Statistical analysis

The actin ACTB gene is taken as an internal reference, and the relative expression change condition of the paphiopediin gene LOR in the sample group relative to the blank control group is analyzed by adopting a 2-delta Ct method. Analysis results are expressed as Mean ± standard deviation SD, and comparisons among groups are performed using t-test statistical analysis, with both tails, p-values <0.05 considered significant differences, and p-values <0.01 considered extremely significant differences.

4 test results

The sample amentoflavone has no up-regulation effect on the LOR expression of the skin keratinocyte paphiopedilum gene, and the gene test results are shown in Table 10.

The test results were as follows: compared with a blank control group, the relative expression quantity of LOR genes in the amentoflavone-0.000156 mg/mL group is 100+/-3%, the p value=0.8044, the difference is not significant, and the amentoflavone-0.000156 mg/mL has no effect of up-regulating the expression of the LOR genes; compared with a blank control group, the relative expression quantity of LOR genes in the amentoflavone-0.000625 mg/mL group is 98+/-8%, the p value= 0.7490, the difference is not significant, and the amentoflavone-0.000625 mg/mL group does not have the effect of up-regulating the expression of the LOR genes; the relative expression quantity of LOR genes in the amentoflavone-0.0025 mg/mL group is 104+/-29%, the p value= 0.9260, the difference is not significant, and the amentoflavone-0.0025 mg/mL has no effect of up-regulating the expression of the LOR genes; the relative expression level of LOR genes in the amentoflavone-0.01 mg/mL group is 115+/-17%, the p value= 0.7274, the difference is not significant, and the amentoflavone-0.01 mg/mL has no effect of up-regulating the expression of the LOR genes.

In conclusion, the quantitative test result of gene expression shows that amentoflavone has no effect of up-regulating the LOR expression of the paphiopedilum gene in skin epidermis keratinocytes within the range of 0.000156-0.01mg/mL.

Table 10: quantitative test results of Gene expression

Application example

The amentoflavone of the present invention can be used as an active substance for the preparation of skin external preparations, preferably cosmetic compositions such as lotions, creams, lotions, eye creams, masks, essences, etc.

The amentoflavone active compound is 0.00001% -10% (w/w), preferably 0.0001% -5% (w/w), more preferably 0.001% -2% (w/w), and most preferably 0.002% -1% (w/w) of the skin external preparation.

The specific application of amentoflavone in skin external preparation, and the formulation and preparation method of these dosage forms are shown in application examples 1-3.

The amentoflavone of the invention can also be used as an active substance for personal care cleaning products, and the personal cleaning products are preferably rinse-off cleaning product compositions, including but not limited to the preparation of products in the forms of body washes for infants, shampoo body washes, body washes for children and adults, shampoo, hand washes, face washes and the like. The plant extracts of this patent are also included in the application of the product form changes, such as powder, cake, paste, bar, tablet, or the like, or intermediates, and the like.

Specific application of amentoflavone in personal cleaning care products, and formulations and preparation methods of these formulations are described in application examples 4-12.

Specific applications are as follows:

application example 1: preparation of toning lotion

Application example 2: preparation of face cream

Application example 3: preparation of the emulsion

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Application example 4: preparation of bath lotion

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Application example 5: preparation of face cleaning emulsion

Application example 6: preparation of bath lotion for infants

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Application example 7: preparation of shampoo for infants

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Application example 8: preparation of shampoo and bath lotion for infants

Application example 9: shampoo preparation

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Application example 10: preparation of hand cleanser

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Application example 11: preparation of scalp care essence

Application example 12: preparation of aerosol (cleaning foam)

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Claims

1. An expression promoter of a small protein gene SPRR2A with rich proline in keratinocytes, wherein the expression promoter is amentoflavone.

2. The expression enhancer of claim 1, wherein the expression enhancer is used at a concentration of at least 0.000156mg/ml.

3. The expression enhancer of claim 2, wherein the expression enhancer is used at a concentration of 0.000156-0.01mg/mL.

4. The expression enhancer of claim 3, wherein the expression enhancer is used at a concentration of 0.000625-0.01mg/mL.

5. The expression promoter according to claim 1, wherein amentoflavone has a purity of 98% by weight or more.

6. The expression promoter according to claim 1, wherein the relative expression level of the keratinocyte-rich proline-rich small protein gene SPRR2A of amentoflavone is not less than 116%.

7. The expression promoter according to claim 6, wherein the relative expression level of the keratinocyte-rich proline-rich small protein gene SPRR2A of amentoflavone is 116-202%.

8. The application of amentoflavone in preparing skin external preparation for promoting expression of proline-rich small protein gene SPRR2A in keratinocyte is provided.

9. The use as claimed in claim 8, wherein the amentoflavone is contained in the external preparation for skin in an amount of 0.00001 to 10% by weight.

10. The use as claimed in claim 8, wherein the amentoflavone is contained in the external preparation for skin in an amount of 0.002 to 1% by weight.

11. The use according to any one of claims 8-10, wherein the skin external agent is selected from the group consisting of: face cream, milky lotion, jelly, pack, face toilet, pack, aerosol cleansing foam, spray, body wash, or facial cleanser.