CN117815091A

CN117815091A - Expression promoter of lipid transport gene ABCA12

Info

Publication number: CN117815091A
Application number: CN202311862745.4A
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 keratinocyte lipid transport gene ABCA12, wherein the expression promoter is amentoflavone. Preferably, amentoflavone is used at a concentration of at least 0.000625mg/ml. The invention also relates to application of amentoflavone in preparing skin external preparation with the effect of promoting expression of keratinocyte lipid transport gene ABCA 12. Preferably, the amentoflavone is contained in the skin external preparation in an amount of 0.00001 to 10 wt%.

Description

Expression promoter of lipid transport gene ABCA12

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 has the function of up-regulating lipid transport gene ABCA12 of skin epidermis keratinocytes, and the amentoflavone can be used as an accelerator for the expression of the lipid transport gene ABCA12 of skin epidermis keratinocytes.

Background

ABC transporters (ATP-binding cassette transporters) are a large superfamily of transporters that bind and hydrolyze ATP and transport various molecules across a restricted biological membrane or into vesicles. ABCA subfamily members are thought to transport lipid substances, ABCA12 being a keratinocyte transmembrane lipid transporter associated with the transport of lipids through lamellar particles. ABCA12 can transport lipids, including ceramides, forming extracellular lipid layers in the stratum corneum, which is critical to skin permeability function. In literature studies, lipid transport by ABCA12 is found to be necessary for keratinocyte differentiation and epidermal morphogenesis. Defective lipid transport due to loss of ABCA12 function results in accumulation of intracellular lipids in epidermal keratinocytes.

Because the ABCA12 transporter is used as a keratinocyte lipid transporter to play a physiological role in keratinocyte differentiation, epidermis morphology and formation of lipid permeability functions, the skin epidermis cell differentiation and formation of lipid permeability functions can be regulated by researching an active substance for regulating and controlling the expression of a lipid transport gene ABCA12, so that the aim of maintaining normal physiology of skin is fulfilled. Therefore, the research of the active substance has positive significance and important application value for promoting the reconstruction of the function of the epidermis of the damaged skin and recovering the normal physiological function of the epidermis of the skin.

The biflavanoid compound is a special flavonoid natural active ingredient extracted from gymnosperms such as ginkgo, selaginella, biota orientalis, selaginella, taxus canadensis and the like, and has various biological activities such as antioxidation, anti-inflammatory, neuroprotection, anticancer, antitumor and the like through modern pharmacological research. Amentoflavone (ambroxol) is a biflavanoid compound extracted from herba Selaginellae (Selaginella tamariscina (Beauv.) Spring) or herba Selaginellae (Selaginella pulvinata (hook. Et Grev.) Maxim.) of Selaginella (Selaginella), and has antioxidant, antiinflammatory, antibacterial, and anticancer effects.

Herba Selaginellae, whole plant of herba Selaginellae of Selaginella (Selaginella) of Pteridaceae, also called herba Corallodisci Cordatae. It is a perennial upright evergreen herb fern plant with the height of 5 cm-15 cm, brown stem, clustered branches, flat shape and light green. The plant has extremely durable drought resistance, and when the plant is drought in weather, the branches are rolled up and contracted into a group so as to keep the moisture in the body; once the rainwater is moistened, the temperature rises, the contracted branches can be spread out, so the branches are also called as herba Cordyotidis Diffusae, rohdea japonica, herba grown grass and the like. The long-term clinical practice and modern pharmacological verification of the traditional medicine in China prove that the traditional Chinese medicine has the effects of clearing heat and detoxicating, activating blood and dissolving stasis, stopping bleeding and resisting cancer, preventing and treating cancer, resisting inflammation and virus, resisting infection, resisting allergy, easing pain, hypnosis, reducing blood pressure, reducing blood sugar, enhancing the immune function of a human body and the like.

The selaginella tamariscina has abundant medicinal experience and various clinical effects, and pharmacological effect researches on amentoflavone are more, but researches and patents on the aspects of promoting lipid transport of epidermal cells and regulating skin functions of selaginella tamariscina extract are less.

In the process of researching and controlling the related active substances of the skin epidermal cell differentiation, it is unexpectedly found that amentoflavone has the function of up regulating the expression of a lipid transport gene ABCA12 in a lipid forming cell and has the effects of promoting the lipid transport in an epidermal keratinocyte and improving the epidermal function.

Disclosure of Invention

In one aspect, the invention provides an expression promoter of keratinocyte lipid transport gene ABCA12, wherein the expression promoter is amentoflavone.

In a preferred embodiment, the expression enhancer is used at a concentration of at least 0.000625mg/mL, preferably 0.000625-0.01mg/mL. Preferably, the expression enhancer is used at a concentration of 0.0025 to 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 amentoflavone of the keratinocyte lipid transport promoting gene ABCA12 is greater than or equal to 118%, preferably from 118 to 159%.

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 keratinocyte lipid transport gene ABCA 12.

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 functions of increasing the expression of lipid transport gene ABCA12 in the lipid forming cells, promoting the differentiation of epidermal keratinocytes and improving the epidermal functions.

The invention has the beneficial effects that the natural compound amentoflavone derived from the Chinese herba selaginellae is found and can be used as an accelerator of lipid transport gene ABCA12 gene in keratinocytes, has the effects of accelerating lipid transport of epidermal keratinocytes and improving 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 epidermis of the skin.

Amentoflavone from amentoflavone

The present invention surprisingly found that amentoflavone has an effect of upregulating expression of the lipid transport gene ABCA12 in cytoplasmic cells. Therefore, amentoflavone can be used as an accelerator for lipid transport gene ABCA12 expression in keratinocytes, applied to development and application of various cosmetic products, and endowed with the effects and functions of reconstructing the function of the epidermis of the skin and recovering the normal physiological function 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 lipid transport gene ABCA 12. In some embodiments, amentoflavone is used at a concentration of at least 0.000625mg/ml.

In some embodiments, amentoflavone is used at a concentration of 0.000625-0.01mg/ml, preferably 0.0025-0.01mg/ml.

In some embodiments, amentoflavone is preferably used at a concentration of less than 0.0127mg/ml, more preferably 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 selaginella water extract extractum, and analyzes the maximum nontoxic dose of the amentoflavone and selaginella water extract extractum 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.).

The preparation method of the selaginella water extract comprises the following steps: extracting active ingredients of herba Selaginellae by double distilled water heating reflux method, and concentrating under reduced pressure to obtain herba Selaginellae water extract.

-medicinal material and instrument

Medicinal materials: the crude drug Selaginella tamariscina is purchased in 5 months 2023 in the market of Chinese medicinal materials in Sichuan lotus pool, and is dried whole herb of Spring of Selaginella tamariscina Selaginella tamariscina (Beauv.) belonging to Selaginella.

Experimental instrument: 450 type pulverizer is purchased from Shandong Moke powder technology equipment Co., ltd, and 200L of extraction and concentration equipment Y-TN-200L is purchased from Shanghai Yu inkstone mechanical equipment Co., ltd.

Preparation of water extract of selaginella

1) Sun-drying herba Selaginellae, mechanically pulverizing with pulverizer, sieving with 100 mesh sieve, and oven drying.

2) Accurately weighing 9.5kg of powder sample, placing into an extraction tank, adding double distilled water with a feed-liquid ratio of 1:10 (1 kg of medicinal material 10L solvent), shaking, and soaking thoroughly.

3) Heating and reflux-extracting for 1h in reflux-extracting concentrating equipment at 80deg.C for 1 time.

4) Adding gauze at the mouth of the extraction tank, filtering the residue to a concentration tank, and concentrating under reduced pressure to remove solvent to obtain herba Selaginellae water extract.

5) Split charging and weighing are carried out to obtain 1.88kg of water extract (see table 1) in total, wherein the water extract of selaginella is a tan paste substance.

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 (tables 1 and 2). 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

Table 2: concentration of herba Selaginellae water extract

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 calculation of the formula,

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

The results of the amentoflavone cytotoxicity test are shown in Table 3, and the results of the Selaginella tamariscina water extract cytotoxicity test are shown in Table 4.

The calculation and analysis are carried out according to the maximum nontoxic dose when the cell activity value is 70%, the maximum nontoxic dose of amentoflavone in keratinocyte treatment is 0.0127mg/mL, and the maximum nontoxic dose of herba Selaginellae water extract in keratinocyte treatment is 1.855mg/mL.

Table 3: amentoflavone cytotoxicity test results

Table 4: cytotoxicity test result of herba Selaginellae water extract

Example 2: abies spicata biflavone regulation ABCA12 gene test

The invention adopts a quantitative detection method of gene expression to analyze the regulation and control condition of amentoflavone on the keratinocyte ABCA12 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 a lipid transport gene ABCA12 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 permeability function or not 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 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.

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. 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 5.

Table 5: 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 6, and the primers used for quantitative detection are shown in Table 7.

Table 6: 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 7: primer sequence for quantitative detection of gene expression

3. Statistical analysis

The relative expression change of the lipid transport gene ABCA12 in the sample group relative to the blank control group is analyzed by adopting a 2-delta Ct method by taking the actin ACTB gene as an internal reference. 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-regulating effect on the expression of the lipid transport gene ABCA12 of the skin epidermis keratinocyte, and the gene test results are shown in Table 6.

The test results were as follows: compared with a blank control group, the relative expression quantity of the ABCA12 genes in the amentoflavone-0.000156 mg/mL group is 112+/-3%, the p value=0.0500, the difference is not significant, and the amentoflavone-0.000156 mg/mL does not have the effect of up-regulating the expression of the ABCA12 genes; the relative expression quantity of ABCA12 genes in the amentoflavone-0.000625 mg/mL group is 118+/-1%, 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 ABCA12 genes; the relative expression quantity of ABCA12 genes in the amentoflavone-0.0025 mg/mL group is 142+/-7%, 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 ABCA12 genes; the relative expression quantity of ABCA12 genes in the amentoflavone-0.01 mg/mL group is 159+/-15%, the p value is less than 0.01, the difference is extremely remarkable, and the amentoflavone-0.01 mg/mL has the function of up-regulating the expression of the ABCA12 genes.

In conclusion, the quantitative test result of gene expression shows that amentoflavone has the function of up-regulating the expression of lipid transport gene ABCA12 in lipid forming cells within the range of 0.000625-0.01mg/mL, and has the effects of promoting the differentiation of epidermal keratinocytes and improving the permeability of epidermis.

Table 8: quantitative test results of Gene expression

Comparative example 1: ABCA12 gene test for regulating and controlling water extract of selaginella

The invention adopts a quantitative detection method of gene expression to analyze the regulation and control condition of the ABCA12 gene of the keratinocytes in the selaginella water extract prepared in the example 1.

1. Cell, reagent consumable and instrument

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 9.

Table 9: reverse transcription system

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 10, and the primers used for quantitative detection are shown in Table 11.

Table 10: fluorescent real-time quantitative polymerase chain reaction system

Table 11: primer sequence for quantitative detection of gene expression

3 statistical analysis

5.4 test results

The water extract of herba Selaginellae has no up-regulating effect on lipid transport gene ABCA12 expression of skin keratinocyte, and the gene test results are shown in Table 12.

The test results were as follows: compared with a blank control group, the relative expression quantity of the ABCA12 gene in the water extract of selaginella tamariscina-0.0625 mg/mL group is 95+/-8%, the p value is= 0.3498, the difference is not significant, and the water extract of selaginella tamariscina-0.0625 mg/mL does not have the effect of up-regulating the expression of the ABCA12 gene; compared with a blank control group, the relative expression quantity of the ABCA12 gene in the water extract of selaginella tamariscina-0.125 mg/mL group is 93+/-6%, the p value=0.1849, the difference is not significant, and the water extract of selaginella tamariscina-0.125 mg/mL has no effect of up-regulating the expression of the ABCA12 gene; the relative expression quantity of the ABCA12 gene in the water extract of selaginella tamariscina-0.25 mg/mL group is 102+/-5%, the p value= 0.7201, the difference is not significant, and the water extract of selaginella tamariscina-0.25 mg/mL has no effect of up-regulating the expression of the ABCA12 gene; the relative expression quantity of the ABCA12 gene in the water extract of selaginella tamariscina-0.5 mg/mL group is 92+/-3%, the p value= 0.1372, the difference is not significant, and the water extract of selaginella tamariscina-0.5 mg/mL has no effect of up-regulating the expression of the ABCA12 gene; the relative expression quantity of the ABCA12 gene in the water extract of selaginella tamariscina-1.0 mg/mL group is 111+/-3%, the p value=0.0729, the difference is not significant, and the water extract of selaginella tamariscina-1.0 mg/mL has no effect of up-regulating the expression of the ABCA12 gene.

In conclusion, the quantitative test result of gene expression shows that the selaginella water extract has no effect of up-regulating the expression of lipid transport gene ABCA12 in skin epidermis keratinocytes in the range of 0.0625-1.0 mg/mL.

Table 12: quantitative test results of Gene expression

Comparative analysis of example 2 and comparative example 1

Example 2 quantitative test of Gene expression shows that amentoflavone has an effect of up-regulating the expression of the lipid transport gene ABCA12 in cytoplasmic forming cells in the range of 0.000625-0.01 mg/mL; comparative example 1 quantitative test results of gene expression show that the water extract of selaginella tamariscina does not have the effect of up-regulating the expression of lipid transport gene ABCA12 in skin epidermis keratinocytes in the range of 0.0625-1.0 mg/mL. This indicates that the ability of amentoflavone in example 2 to upregulate lipid transport gene ABCA12 expression in lipid forming cells is superior to that of the water extract of selaginella tamariscina in comparative example 1.

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

Application example 4: preparation of bath lotion

Application example 5: preparation of face cleaning emulsion

Application example 6: preparation of bath lotion for infants

Application example 7: preparation of shampoo for infants

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

Application example 9: shampoo preparation

Application example 10: preparation of hand cleanser

Application example 11: preparation of scalp care essence

Application example 12: preparation of aerosol (cleaning foam)

Claims

1. An expression promoter of keratinocyte lipid transport gene ABCA12, wherein said expression promoter is amentoflavone.

2. The expression enhancer of claim 1, wherein the expression enhancer is used at a concentration of at least 0.000625mg/ml.

3. The expression enhancer of claim 2, wherein the expression enhancer is used at a concentration of 0.000625-0.01mg/mL.

4. The expression enhancer of claim 3, wherein the expression enhancer is used at a concentration of 0.0025 to 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 lipid transport promoting gene ABCA12 of amentoflavone is not less than 118%.

7. The expression promoter according to claim 5, wherein the relative expression level of the keratinocyte lipid transport promoting gene ABCA12 of amentoflavone is 118 to 159%.

8. Use of amentoflavone in the preparation of a skin external preparation having the effect of promoting expression of keratinocyte lipid transport gene ABCA 12.

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.