Beauty application of methylophiopogonin A

Abstract

The invention provides a cosmetic application of methylophiopogonin A. The cosmetic application is selected from the group consisting of promotion of IV-type collagen, promotion of VII-type collagen or a combination of the IV-type collagen and the VII-type collagen. The invention also relates to an application of the methylophiopogon japonicus flavone A in preparation of a skin external preparation with an effect of promoting generation of type IV collagen and / or type VII collagen.

Description

Technical Field

[0001] The present invention relates to the research on the cosmetic effect of methyl ophiopogon japonicus flavonoids A, and specifically to the application of methyl ophiopogon japonicus flavonoids A in promoting the production of type IV collagen and / or type VII collagen, and the application of methyl ophiopogon japonicus flavonoids A in the preparation of a skin external preparation having the effect of promoting the production of type IV collagen and / or type VII collagen. Background Art

[0002] Collagen is a biopolymer, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein, and even up to 80% or more in some organisms. There are many types of collagen, the common types are: Type I (dermis, bones, tendons, ligaments, etc.), Type II (cartilage, vitreous, etc.), Type III (skin, blood vessels, intestines, etc.), Type IV (base membrane, etc.), Type V (bone, dermis, cornea, placenta, etc.). Collagen has good biocompatibility, biodegradability and biological activity, so it is widely used in food, medicine, tissue engineering, cosmetics and other fields.

[0003] Type IV collagen is the most abundant collagen fiber in the dense layer of the basement membrane. It constitutes the vast majority of the dense layer and is an important supporting structure of the basement membrane. After being synthesized in the cell, it directly participates in the formation of the extracellular matrix as procollagen. Serum type IV collagen is considered to reflect the synthesis of collagen and is positively correlated with the fiber process.

[0004] Type VII collagen is a genetically unique member of the collagen family. It is the main component of anchoring fibrils, which are complexes that attach the basement membrane of the skin to the dermis below. Type VII collagen is mainly distributed in the dense lower layer. Anchoring fibrils are stable dimers of type VII collagen bound by disulfide bonds. Type VII collagen binds to the laminin 5-6 / 7 complex through nestin to form a collagen network. Type VII collagen binds to the basement membrane through an interaction with type IV collagen.

[0005] Methylophiopogonone A, CAS No.: 74805-90-6, English name: Methylophiopogonone a, molecular formula: C 19 H 16 O6, molecular weight: 340.327, structural formula is as follows:

[0006]

[0007] At present, there are few studies on the efficacy of methyl ophiopogon japonicus flavonoids A. In patent CN 112294794, it was found that methyl ophiopogon japonicus flavonoids A is an nrf2 agonist, and nrf2 is a key regulatory factor in anti-oxidative stress and anti-inflammatory response; in patent JP 2004-143073, it was found that methyl ophiopogon japonicus flavonoids A can inhibit the activity of tyrosinase. However, to date, there has been no report that methyl ophiopogon japonicus flavonoids A has the effect of promoting the production of type IV collagen and / or type VII collagen.

[0008] This study surprisingly found that methyl ophiopogon japonicus flavonoid A can promote the proliferation of fibroblast collagen IV and collagen VII, and can be used as an effective additive to prepare skin care products to help improve related problems. Summary of the invention

[0009] The present invention provides a cosmetic application of methyl ophiopogon japonicus flavonoids A, wherein the cosmetic application is selected from: promoting type IV collagen, promoting type VII collagen or a combination thereof.

[0010] In a preferred embodiment, the concentration of the methyl ophiopogon japonicus flavonoids A is at least 0.005 mg / mL. In a preferred embodiment, the concentration of the methyl ophiopogon japonicus flavonoids A is below 0.01 mg / mL.

[0011] In a preferred embodiment, the type IV collagen and type VII collagen are derived from fibroblasts.

[0012] On the other hand, the present invention also relates to the use of methyl ophiopogon japonicus flavonoids A in the preparation of a skin external preparation having the effect of promoting the production of type IV collagen and / or type VII collagen.

[0013] In a preferred embodiment, the content of methyl ophiopogon japonicus flavonoids A in the external skin preparation is 0.0000001-20% by weight, more preferably 0.0000001-1% by weight.

[0014] In a preferred embodiment, the skin external preparation is selected from: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol cleansing foam, spray, shower gel, or facial cleanser. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shown are the results of the MTT assay for fibroblast viability. DETAILED DESCRIPTION

[0017] The present invention relates to the effect of methyl ophiopogon japonicus flavonoids A on promoting the production of type IV collagen and / or type VII collagen. It is discovered for the first time that methyl ophiopogon japonicus flavonoids A can be used as a functional additive in skin topical preparations to help achieve the effects of maintaining skin firmness, providing support and stability for surrounding tissues, improving skin sagging and / or wrinkles, and resisting skin aging.

[0018] The lipopolysaccharide A described herein may optionally be in the form of finished product packaging. In one embodiment, the packaging is a container such as a plastic, metal or glass tube or a wide-mouthed bottle containing lipopolysaccharide A. The product may additionally have a packaging such as a plastic or cardboard box for storing the container. In one embodiment, the product comprises lipopolysaccharide A and has instructions for guiding the user to apply lipopolysaccharide A to the skin to treat signs of skinization as discussed below. Such instructions may be printed on a container, a label insert or any other packaging.

[0019] As used herein, "topical application" means applying or spreading directly onto the external skin, scalp, or hair, for example, using the hands or an application device such as a wipe, roller, or spray.

[0020] As used herein, "cosmetic" refers to cosmetic substances or preparations that maintain, restore, impart, stimulate, or enhance the physical appearance or seem to increase beauty or youthfulness, especially as it relates to the appearance of tissue or skin.

[0021] As used herein, "cosmetically effective amount" means an amount of a physiologically active compound or composition sufficient to treat one or more skin conditions, but low enough to avoid serious side effects. The cosmetically effective amount of a compound or composition will vary depending on the specific condition being treated, the age and physical condition of the end user, the severity of the condition being treated / prevented, the duration of the treatment, the nature of other treatments, the specific compound or product / composition employed, the specific cosmetically acceptable carrier utilized, and the like.

[0022] In order to provide a more concise description, some quantitative expressions given herein are not modified by the term "about". It should be understood that, whether or not the term "about" is explicitly used, each quantity given herein is intended to refer to the actual given value, and is also intended to refer to the approximate values ​​of these given values ​​that can be reasonably inferred by ordinary technicians in the field, including the approximate values ​​of these given values ​​caused by experimental and / or measurement conditions.

[0023] To provide a more concise description, some quantitative expressions herein are described as a range of about X amount to about Y amount. It should be understood that when describing a range, the range is not limited to the upper and lower limits described, but should include the entire range of about X amount to about Y amount or any amount therebetween.

[0024] Methyl ophiopogon japonicus flavonoids A

[0025] The present invention is based on the following unexpected discovery: methyl ophiopogon japonicus flavonoids A has the effect of promoting the production of type IV collagen and / or type VII collagen. Therefore, methyl ophiopogon japonicus flavonoids A can be added to skin care products as an effective ingredient to help improve skin sagging, wrinkle problems, etc. The purpose of the present invention is to provide the effect of methyl ophiopogon japonicus flavonoids A in promoting the production of type IV collagen and / or type VII collagen and its application in cosmetics.

[0026] In some embodiments, the concentration of methyl ophiopogon japonicus A is at least about 0.005 mg / mL. In a preferred embodiment, the concentration of methyl ophiopogon japonicus A is below about 0.01 mg / mL. In some embodiments, the concentration of methyl ophiopogon japonicus A is about 0.005-0.01 mg / mL.

[0027] Skin topical products

[0028] In some embodiments, methyl ophiopogon japonicus flavonoids A can be used for the preparation of skin external preparations. The skin external preparations are preferably cosmetic compositions, including but not limited to products in dosage forms such as creams, lotions, gels, toners, essences, masks, eye creams, aerosols (cleansing bubbles), sprays, shower gels, and facial cleansers.

[0029] The skin external preparation is a general concept for all ingredients usually used on the outside of the skin, for example, it can be a cosmetic composition. The cosmetic composition can be a basic cosmetic, a facial makeup cosmetic, a body cosmetic, a hair care cosmetic, etc., and there is no special restriction on its dosage form, which can be reasonably selected according to different purposes. The cosmetic composition also contains different media or matrix excipients allowed by the cosmetics level according to different dosage forms and purposes.

[0030] The skin external preparation comprising methyl ophiopogon japonicus flavonoids A can be topically applied to human skin and / or hair. The skin external preparation can also include a cosmetically acceptable topical carrier, which can be about 50 wt % to about 99.99 wt % of the skin external preparation (such as about 80 wt % to about 99 wt % of the skin external preparation). In a preferred embodiment of the present invention, the cosmetically acceptable topical carrier comprises water. The cosmetically acceptable topical carrier may include one or more selected from wetting agents, emollients, grease, wetting agents and similar substances. In one embodiment, the cosmetically acceptable topical carrier comprises a substrate such as a nonwoven fabric or a membrane material.

[0031] Skin topical agents can be prepared 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, shaving creams, wipes, patches, hydrogels, film-forming products, facial masks and skin films, films and cosmetics such as foundation and mascara. These product types can 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.

[0032] The skin external preparation containing methyl ophiopogon japonicus flavonoid A can be formulated as a solution. The solution generally contains an aqueous solvent or an organic solvent (e.g., about 50% to about 99.99% or about 90% to about 99% of a cosmetically acceptable aqueous solvent 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.

[0033] Skin topical preparations can be formulated into solutions containing emollients. Such skin topical preparations preferably contain from about 2% to about 50% of one or more emollients. As used herein, "emollient" refers to a substance used to prevent or alleviate 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, etc.

[0034] 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%) water.

[0035] Another type of product that can be formulated from a solution is a cream. Creams typically contain 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%) water.

[0036] Although it is preferred that the skin external preparation containing methyl ophiopogon japonicus A contains water, as another option, the skin external preparation can be anhydrous or an ointment containing no water but organic and / or silicone solvents, greases, lipids and waxes. The ointment can contain a simple base of animal or vegetable oil or semisolid hydrocarbon. The ointment can contain about 2% to about 10% of one or more emollients and about 0.1% to about 2% of one or more thickeners.

[0037] The topical agent for skin application can be formulated as an emulsion. If the topical carrier is an emulsion, about 1% to about 10% (e.g., about 2% to about 5%) of the topical carrier contains one or more emulsifiers. The emulsifier can be nonionic, anionic or cationic. Examples of suitable emulsifiers include those commonly identified as suitable emulsifiers in the field of personal care and cosmetic formulations.

[0038] Lotions and creams can be formulated as emulsions. Typically such lotions contain 0.5% to about 5% of one or more emulsifiers. Such creams typically contain about 1% to about 20% (e.g., about 5% to about 10%) of one or more emollients; about 20% to about 80% (e.g., 30% to about 70%) of water; and about 1% to about 10% (e.g., about 2% to about 5%) of one or more emulsifiers.

[0039] Oil-in-water and water-in-oil type single emulsion skin care preparations, such as lotions and creams, are well known in the cosmetics field and can be used in the present invention. Multiphase emulsion skin external preparations (such as water-in-oil-in-water type and oil-in-water-in-oil type) can also be used in the present invention. Generally, such single-phase emulsions or multiphase emulsions contain water, emollients and emulsifiers as their basic ingredients.

[0040] The skin external preparation comprising methyl ophiopogon japonicus flavonoids A can also be formulated into a gel (e.g., an aqueous gel, an alcohol gel, an alcohol / water type gel or an oily gel using a suitable gelling agent). Suitable gelling agents for aqueous gels and / or alcohol 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 gelling agents for oils (e.g., mineral oils) include, but are not limited to, hydrogenated butylene / ethylene / styrene copolymers and hydrogenated ethylene / propylene / styrene copolymers. Such gelling agents typically contain between about 0.1% by weight and 5% by weight of such gelling agents.

[0041] The external preparation for skin application comprising ophiopogon japonicus A can also be formulated into a solid preparation (eg, a wax-based stick, a bar soap, a powder, or a wipe containing the powder).

[0042] In addition to the above-mentioned components, the external preparation for skin which can be used in the present invention may contain various other oil-soluble substances and / or water-soluble substances at levels conventionally established in its technical field for use in external preparations for skin for use on skin and hair.

[0043] The skin external preparation of the present invention may contain additional components commonly found in skin care compositions, such as moisturizers, skin regulators, emulsifiers, preservatives, antioxidants, fragrances, chelating agents, etc., as long as they are physically and chemically compatible with other components in the skin external preparation and do not affect the effects of the methyl ophiopogon japonicus flavonoids A of the present invention.

[0044] In some embodiments of the skin topical preparation of the present invention, one or more preservatives may be used. Suitable preservatives include p-hydroxyacetophenone, C1-C4 alkyl parahydroxybenzoate and phenoxyethanol. Based on the total weight of the composition, the amount of the preservative is about 0.5 to about 2 weight %, preferably about 0.5 to 1 weight %.

[0045] In one example of the skin external preparation of the present invention, one or more antioxidants can be used. Suitable antioxidants include butylated hydroxytoluene (BHT), ascorbyl palmitate (BHA), butylated hydroxyanisole, phenyl-α-naphthylamine, hydroquinone, propyl gallate, nordihydroguaiaretic acid, vitamin E or vitamin E derivatives, vitamin C and its derivatives, calcium pantothenate, green tea extract and mixed polyphenols, and mixtures of the above-mentioned substances. The antioxidant used is about 0.02 to 0.5 weight % of the total weight of the composition, and more preferably from about 0.002 to 0.1 weight % of the amount used.

[0046] In one example of the skin external preparation of the present invention, one or more emollients can be used to act as lubricants through their ability to remain on the skin surface or in the stratum corneum to reduce flaking and improve the appearance of the skin. Typical emollients include fatty esters, fatty alcohols, mineral oils, polyether silicone copolymers, and the like. Examples of suitable emollients include, but are not limited to, polypropylene glycol ("PPG")-15 stearyl ether, PPG-10 cetyl ether, Steareth-10, Oleth-8, PPG-4 lauryl ether, vitamin E acetate, lanolin, cetyl alcohol, cetearyl ethylhexanoate, cetearyl alcohol, glyceryl stearate, hydroxystearate octyl, dimethyl polysiloxane, and combinations thereof. Cetyl alcohol, cetearyl ethylhexanoate, cetearyl alcohol, glyceryl stearate, and combinations thereof are preferred. When used, the emollient is used in an amount ranging from about 0.1 to about 30% by weight, preferably about 1 to about 30% by weight, based on the total weight of the composition.

[0047] In one example of the skin external preparation of the present invention, one or more moisturizers can be used. Moisturizers are also called humectants, which help to improve the effect of emollients, reduce flaking, remove the scales of the composition and improve the touch of the skin. Polyols can be used as moisturizers, including but not limited to glycerol, 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 glycerol, propoxylated glycerol, and combinations thereof. When used, the amount of moisturizer is about 0.1 to about 20% by weight, preferably about 1 to about 15% by weight, based on the total weight of the composition.

[0048] In an example of the skin external preparation of the present invention, one or more emulsifiers can be used. Emulsifiers can be used within the scope of effective stabilizing amount. Preferably, based on the total weight of the composition, the emulsifier is used in an amount of about 1.0 to about 10.0 weight %, more preferably about 3.0 to about 6.0 weight %. Any emulsifier compatible with the components in the composition can be used. Suitable emulsifiers include stearic acid, cetyl alcohol, glyceryl stearate, lecithin, stearyl alcohol, Steareth-2, Steareth-20, acrylic acid (ester) class / C10-30 alkyl alcohol acrylate cross-linked polymer, and combinations thereof.

[0049] In one example of the skin external preparation of the present invention, one or more pH regulators can be used. The pH regulator useful in the skin external preparation of the present invention includes tromethamine. When used, the amount of the pH regulator based on the total weight of the composition is about 0.1 to about 2 weight %, preferably about 0.1 to about 1 weight %.

[0050] In a specific embodiment of the present invention, the skin topical agent comprises acrylates / C10-30 alkyl acrylate crosspolymer, glycerin, p-hydroxyacetophenone, glyceryl stearate and lecithin, cetearyl alcohol, cetearyl ethylhexanoate, tromethamine or a combination thereof.

[0051] Additional cosmetic active agents

[0052] In some embodiments, the topical skin preparation may also include additional cosmetic active agents. As used herein, "cosmetic active agents" are compounds (such as synthetic compounds or compounds isolated from natural sources or natural extracts) that have cosmetic or therapeutic effects on skin or hair, including but not limited to anti-acne agents, oil control agents, antimicrobial agents, anti-inflammatory agents, antifungal agents, antiparasitic agents, topical analgesics, sunscreens, photoprotectants, antioxidants, keratolytic agents, surfactants, moisturizers, nutrients, vitamins, energy enhancers, antiperspirants, astringents, deodorants, curing agents, anti-hard cuticle agents, and agents for hair and / or skin conditioning.

[0053] In one embodiment, these cosmetic actives are selected from (but not limited to): hydroxy acids, benzoyl peroxide, D-panthenol, octyl methoxycinnamate, titanium dioxide, octyl salicylate, homosalate, avobenzone, carotenoids, free radical scavengers, spin traps, amines, retinoids such as retinol and retinyl palmitate, ceramides, polyunsaturated fatty acids, essential fatty acids, enzymes, enzyme inhibitors, minerals, hormones such as estrogen, steroids such as hydrocortisone, 2-dimethylaminoethanol, copper salts such as copper chloride, copper-containing peptides such as Cu:Gly-His-Lys, coenzyme Q10, peptides, amino acids such as proline, vitamins, lactobionic acid, acetyl-CoA, niacin, riboflavin, thiamine, ribose, electron transfer substances such as NADH and FADH2, and other plant extracts such as aloe, feverfew, oatmeal, and their derivatives and mixtures. The cosmetic active agent is generally present in an amount of about 0.001% to about 20%, for example about 0.005% to about 10%, such as about 0.01% to about 5%, by weight of the topical skin preparation of the present invention.

[0054] Examples of vitamins include, but are not limited to, vitamin A, vitamin B (e.g., vitamin B3, vitamin B5, and vitamin B12), vitamin C, vitamin K, and different forms of vitamin E (e.g., α, β, γ, or Δ tocopherol) or mixtures thereof and derivatives thereof.

[0055] Examples of hydroxy acids include, but are not limited to, glycolic acid, lactic acid, malic acid, salicylic acid, citric acid, and tartaric acid.

[0056] Examples of antioxidants include, but are not limited to, water-soluble antioxidants such as sulfhydryl compounds and their derivatives (e.g., sodium metabisulfite and N-acetyl-cysteine), lipoic acid and dihydrolipoic acid, resveratrol, lactoferrin and ascorbic acid and ascorbic acid derivatives (e.g., ascorbyl palmitate and ascorbic acid polypeptide). Oil-soluble antioxidants suitable for use in the skin topical preparations of the present invention include, but are not limited to, butylated hydroxytoluene, retinoids (e.g., retinol and retinyl palmitate), tocopherols (e.g., tocopherol ethyl ester), tocotrienols and ubiquinone. Natural extracts containing antioxidants suitable for use in the skin topical preparations of the present invention include, but are not limited to, extracts containing flavonoids and isoflavones and their derivatives (e.g., genistein and di-zein), extracts containing resveratrol, and the like. Examples of such natural extracts include grape seeds, green tea, pine bark, and propolis.

[0057] Example

[0058] The present invention is further described below in conjunction with specific examples. It is necessary to point out that the examples are only used to further illustrate the present invention and cannot be construed as limiting the scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments based on the content of the present invention described above. The test methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, all percentages and parts are by weight.

[0059] The methyl ophiopogon japonicus flavonoids A used in the present invention is purchased from Pufei De, with the item number: JOT-11604.

[0060] Example 1: Sample preparation

[0061] Take methyl ophiopogon japonicus flavonoids A, use DMEM low-glucose culture medium (Gibco, 500ml-11885084), and prepare relevant samples based on fibroblast test, as shown in Table 1.

[0062] Table 1: Sample information of Example 1 based on fibroblast-related experiments

[0063]

[0064] Test Example 1: MTT assay to detect fibroblast viability

[0065] Skin fibroblasts (Guangdong Boxi Biotechnology Co., Ltd., Fb19052002) were cultured at 8×10 3 The cells were inoculated into a 96-well plate at a density of 100 cells / well and cultured in DMEM low-glucose medium (Gibco, 500ml-11885084) + 10% NewbormCalf serum (SERANA, NCS-CN500) + 1% antibiotic-antimycotic (triple antibody), 100X (Shanghai Yuanpei, S120JV).

[0066] The experiment set up a zeroing group, a solvent control group (SC) and a sample group (samples 1-5, 8-10 in Example 1). Three replicate wells were set for each sample. When the plating rate reached about 60%, fibroblasts were inoculated into a 96-well plate and incubated. When the plating rate reached 50% to 60%, the drug was administered. 200 μL of culture solution was added to each well of the SC group; 200 μL of culture solution containing the sample to be tested at the corresponding concentration was added to each well of the sample group; the zeroing group had no cell inoculation and only 200 μL of cell culture solution was added. After the drug administration was completed, the culture was continued. After 24 hours, the supernatant was discarded, and MTT working solution (0.5 mg / mL) was added. The plates were incubated at 37°C in the dark for 4 hours. After the incubation, the supernatant was discarded from each well and DMSO was added. The OD value was read at 490 nm.

[0067] Relative cell viability (%) = (OD 样品孔 -OD 调零孔 ) / (OD 溶剂对照孔 -OD 调零孔 )*100%

[0068] Table 2: Fibroblast viability test results

[0069]

[0070] Figure 1 Shown are the results of MTT assay for fibroblast viability.

[0071] According to Table 2 and Figure 1 The cell viability test results showed that methyl ophiopogon japonicus flavonoids A did not show obvious cytotoxicity within the concentration range of 0.01 mg / mL (sample 9) based on fibroblasts.

[0072] Test Example 2: Detection of Collagen IV gene changes in fibroblasts by fluorescence quantitative PCR

[0073] Skin fibroblasts (Guangdong Boxi Biotechnology Co., Ltd., Fb19052002) were cultured at 2×10 5 Cells were inoculated into 6-well plates at a seeding density of 100 cells / well and cultured using DMEM low-glucose medium (Gibco, 500ml-11885084). "RNAiso Plus" kit (Takara, 9108).

[0074] This test is based on fibroblasts, and detects the changes in the expression of the Collagen IV gene after the action of the example. Set up a blank control group (BC), a positive control group (PC, TGF-β1), and a sample group (samples 7-9 in Example 1). Inoculate cells into a 6-well plate and incubate overnight in an incubator (37°C, 5% CO2). When the cell plating rate reaches 40% to 60%, group medication is carried out, with a dosage of 2.0 mL per well. Each group has 3 replicate wells and is incubated in an incubator (37°C, 5% CO2) for 24 hours. Discard the supernatant, wash twice with PBS, add 1.0 mL of RNAiso Plus to each well, blow and lyse the cells, and collect the samples. Extract RNA, reverse transcribe to cDNA, and perform fluorescent quantitative PCR detection using 2 -△△CT Methods The results were calculated. The results were expressed as Mean ± SD. The t-test was used for statistical analysis among the groups. All statistical analyses were two-tailed. P < 0.05 was considered to be significantly different, expressed as *; P < 0.01 was considered to be extremely significantly different, expressed as **.

[0075] Up-regulation rate (%) = (gene content 样品组-Gene content BC ) / Gene content BC × 100%. The results are shown in Table 3.

[0076] The results of collagen IV content were expressed as Mean±SD (Table 3).

[0077] Table 3: Type IV collagen (Collagen ⅠV) test results

[0078] Experimental Grouping Mean±SD Increase rate BC 1.00±0.01 / PC 2.80±0.02*** 180％ Sample 7 1.08±0.30 / Sample 8 1.35±0.16* 35％ Sample 9 1.26±0.03*** 26％

[0079] As shown in Table 3, compared with the BC group, the expression of Collagen IV gene in the PC group was significantly upregulated, indicating that the positive control of this test was effective. Compared with the BC group, the expression of Collagen IV gene in sample 8 (0.005 mg / mL methyl ophiopogon japonicus flavonoids A) and sample 9 (0.01 mg / mL methyl ophiopogon japonicus flavonoids A) were significantly upregulated, with upregulation rates of 35% and 26%, respectively; the expression of Collagen IV gene in sample 7 (0.0025 mg / mL methyl ophiopogon japonicus flavonoids A) did not change significantly.

[0080] Test Example 3: Detection of Collagen VII gene changes in fibroblasts by fluorescence quantitative PCR

[0081] Skin fibroblasts (Guangdong Boxi Biotechnology Co., Ltd., Fb19052002) were cultured at 2×10 5 Cells were inoculated into 6-well plates at a seeding density of 100 cells / well and cultured using DMEM low-glucose medium (Gibco, 500ml-11885084). "RNAiso Plus" kit (Takara, 9108).

[0082] This test is based on fibroblasts, and detects the changes in the expression of Collagen VII gene after the action of Example 1. Set up a blank control group (BC), a positive control group (PC, TGF-β1), and a sample group (samples 7-9 in Example 1). Inoculate cells into a 6-well plate and incubate overnight in an incubator (37°C, 5% CO2). When the cell plating rate reaches 40% to 60%, group medication is carried out, with a dosage of 2.0 mL per well. Each group has 3 replicate wells and is incubated in an incubator (37°C, 5% CO2) for 24 hours. Discard the supernatant, wash twice with PBS, add 1.0 mL of RNAiso Plus to each well, blow and lyse the cells, and collect the samples. Extract RNA, reverse transcribe to cDNA, and perform fluorescent quantitative PCR detection using 2 -△△CTMethods The results were calculated. The results were expressed as Mean ± SD. The t-test was used for statistical analysis among the groups. All statistical analyses were two-tailed. P < 0.05 was considered to be significantly different, expressed as *; P < 0.01 was considered to be extremely significantly different, expressed as **.

[0083] Up-regulation rate (%) = (gene content 样品组 -Gene content BC ) / Gene content BC × 100%. The results are shown in Table 4.

[0084] The results of collagen VII content were expressed as Mean±SD (Table 4).

[0085] Table 4: Type VII collagen (Collagen VIⅠ) test results

[0086] Experimental Grouping Mean±SD Increase rate BC 1.00±0.16 / PC 3.30±0.07*** 230％ Sample 7 1.01±0.05 / Sample 8 1.30±0.06* 30％ Sample 9 1.20±0.03 20％

[0087] As shown in Table 4, compared with the BC group, the expression of Collagen VII gene in the PC group was significantly upregulated, indicating that the positive control of this test was effective. Compared with the BC group, the expression of Collagen IV gene in sample 8 (0.005 mg / mL methyl ophiopogon japonicus flavonoids A) and sample 9 (0.01 mg / mL methyl ophiopogon japonicus flavonoids A) was upregulated, with the upregulation rates being 30% and 20%, respectively; the expression of Collagen IV gene in sample 7 (0.0025 mg / mL Example 1) had no significant change.

[0088] The methyl ophiopogon japonicus flavonoids A of the present invention can be used as an effective additive for the preparation of medicines, health foods or cosmetics. The skin external preparation is preferably a cosmetic composition, including but not limited to the preparation of products in the form of cream, emulsion, gel, lotion, essence, mask, eye cream, aerosol (cleansing bubble), spray, shower gel and facial cleanser, and the weight percentage of methyl ophiopogon japonicus flavonoids A in the skin external preparation is 0.0000001%-20% (w / w). The preferred weight percentage is 0.000001%-10% (w / w). The more preferred weight percentage is 0.00001%-1% (w / w). The most preferred weight percentage is 0.00001%-0.1% (w / w).

[0089] The following are specific application examples of methyl ophiopogon japonicus flavonoids A in skin topical preparations, as well as the formulations and preparation methods of these dosage forms. The specific application examples are as follows:

[0090] Application Example 1: Preparation of facial cream

[0091]

[0092] Application Example 2: Preparation of Emulsion

[0093]

[0094] Application Example 3: Preparation of Gel

[0095]

[0096]

[0097] Application Example 4: Preparation of lotion

[0098]

[0099] Application Example 5: Preparation of Essence

[0100]

[0101]

[0102] Application Example 6: Preparation of facial mask

[0103]

[0104]

[0105] Application Example 7: Preparation of Eye Cream

[0106]

[0107] Application Example 8: Preparation of Aerosol (Cleaning Bubble)

[0108]

[0109]

[0110] Application Example 9: Preparation of Spray

[0111]

[0112] Application Example 10: Preparation of Shower Gel

[0113]

[0114]

[0115] Application Example 11: Preparation of facial cleanser

[0116]

Claims

1. A cosmetic application of methyl ophiopogon japonicus flavonoids A, wherein the cosmetic application is selected from: promoting type IV collagen, promoting type VII collagen, or a combination thereof.

2. The use according to claim 1, characterized in that The usage concentration of the methyl ophiopogon japonicus flavonoids A is at least 0.005 mg / mL.

3. The use according to claim 1, characterized in that The usage concentration of the methyl ophiopogon japonicus flavonoids A is below 0.01 mg / mL.

4. The use according to claim 1, characterized in that The type IV collagen and type VII collagen are derived from fibroblasts.

5. Use of methyl ophiopogon japonicus flavonoids A in the preparation of a skin external preparation having the effect of promoting the production of type IV collagen and / or type VII collagen.

6. The use according to claim 5, characterized in that The content of methyl ophiopogon japonicus flavonoids A in the external skin preparation is 0.0000001-20% by weight.

7. The use according to claim 5, characterized in that The content of the methyl ophiopogon japonicus flavonoids A in the external skin preparation is 0.0000001-1% by weight.

8. The use according to claim 5, characterized in that The skin external preparation is selected from: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol cleansing foam, spray, shower gel, or facial cleanser.

Images