CN116327626A

CN116327626A - Application of opal D in promotion of collagen and/or elastin production

Info

Publication number: CN116327626A
Application number: CN202310406772.4A
Authority: CN
Inventors: 孙懿; 张东; 陈默; 赵一帆; 赵亚; 杨岚; 祝乐; 贾海东
Original assignee: Institute of Materia Medica of CAMS; Shanghai Jahwa United Co Ltd
Current assignee: Institute of Materia Medica of CAMS; Shanghai Jahwa United Co Ltd
Priority date: 2023-04-17
Filing date: 2023-04-17
Publication date: 2023-06-27

Abstract

The invention provides an application of euonymus alatus phenol D in promoting collagen and/or elastin production. The invention also relates to application of the euonymus alatus phenol D in preparing external skin preparations, medicines and health-care foods with the function of promoting the generation of collagen and/or elastin.

Description

Application of opal D in promotion of collagen and/or elastin production

Technical Field

The invention relates to the field of natural pharmaceutical chemistry, in particular to application of euonymus alatus phenol D in promoting collagen and/or elastin generation, and application of euonymus alatus phenol D in preparing skin external preparations, medicines and health-care foods with the function of promoting collagen and/or elastin generation.

Background

Fibroblasts (Fb) are the main cells constituting the dermis layer of the skin, and are the main cells of the dermis layer that produce collagen and other cellular interstitium, and their normal differentiation and proliferation maintain the normal structure and physiological functions of the skin. Fb has strong protein synthesis capability, can synthesize and secrete a large amount of matrix components such as elastin, collagen, glycosaminoglycan, glycoprotein and the like, further generates elastic fibers, collagen fibers and reticular fibers, and secretes various cell repair factors, so that the skin has strong renewal and self-repair capability, and substances with increased thickness and density of dermis of the skin have potential wrinkle stretching and skin elasticity and luster recovering effects.

The three-dimensional helical structure of collagen supports the skin in the form of springs, and the human body can produce many collagens at younger age, but their yield decreases with age. Collagen (Collagen) can be divided into fibrous Collagen and non-fibrous Collagen. The fiber collagen is mainly I type, III type and V type. Among them, the Collagen I content is most abundant, and has strong tensile strength in relation to skin stretch resistance. Elastin (Elastin) is the major component of elastane fiber. Is particularly abundant in skin connective tissue, exists with collagen fibers, and imparts elasticity and tension to the tissue. The skin can have the ability of stretching and folding, and is responsible for maintaining and supporting the elasticity of the skin. With age, the collagen and elastin content in the skin decreases, thereby reducing the network support required to maintain a firm skin, the skin begins to relax, and fine lines and the like appear.

The opal D is also called 3,6, 7-trimethyl quercetin marigold, and has the English name Chrysosplenol D and CAS number 14965-20-9, and the molecular weight is as follows: 360.318, molecular formula: c (C) ₁₈ H ₁₆ O ₈ . The structural formula of the opal D is as follows:

the herba Euphorbiae Humifusae phenol D is a methoxy flavonoid compound, and exists in fructus Vitics Simplicifoliae, herba Artemisiae Annuae, herba Centipedae, herba Rabdosiae Lophanthoidis, etc. Meanwhile, the compound is the flavonoid compound with the highest content in the sweet wormwood herb. In vitro activity shows that the euonymus alatus phenol D has selective synergistic effect on the antimalarial effect of artemisinin; in vitro researches show that the opal D can induce cell cycle aberration and also can induce breast cancer apoptosis; in vivo studies have reported that eugenol D has protective effects on LPS-induced Systemic Inflammatory Response Syndrome (SIRS) in mice. At present, the research on the opal D is mostly focused on the fields of malaria resistance, inflammation resistance and apoptosis, and the opal D is rarely reported in the skin field.

The invention surprisingly discovers that the opal D has excellent functions of promoting Fb proliferation and promoting collagen and/or elastin generation, and can be used as an efficacy additive to be applied to skin external preparations, medicines and health-care foods.

Disclosure of Invention

In one aspect, the invention provides the use of euonymus alatus phenol D to promote collagen and/or elastin production.

In a preferred embodiment, the concentration of the use of the eugenol D is at least 0.08. Mu.g/mL, preferably at least 0.1. Mu.g/mL.

In a preferred embodiment, the collagen is type I collagen.

On the other hand, the invention also relates to the application of the euonymus alatus phenol D in preparing external skin preparations, medicines and health-care foods with the function of promoting the generation of collagen and/or elastin.

In a preferred embodiment, the content of the eugenol D in the skin external preparation, the medicine and the health food is 0.000008 to 20% by weight, preferably 0.00001 to 10% 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.

Brief description of the drawings

Fig. 1 shows a fibroblast morphology map of the solvent control group.

FIG. 2 shows a fibroblast morphology of 0.9 μg/mL opal D.

FIG. 3 shows a fibroblast morphology of 0.45 μg/mL opal D.

FIG. 4 shows a fibroblast morphology of 0.225 μg/mL opal D.

FIG. 5 shows a fibroblast morphology of 0.1125 μg/mL opal D.

FIG. 6 shows a morphology of fibroblasts for the 0.05625 μg/mL opal D.

Detailed Description

The invention relates to the effect of the opal D in promoting the generation of collagen and/or elastin, and discovers that the opal D can be used as an efficacy additive for preparing external skin preparations, medicines and health-care foods for the first time so as to help to realize the effects of maintaining skin tightness, improving skin relaxation and/or wrinkles, resisting skin aging and the like.

In order to provide a more concise description, some quantitative representations presented herein are not modified by the term "about". It will be understood that each quantity given herein is intended to refer to an actual given value, whether or not the term "about" is explicitly used, and is also intended to refer to approximations of such given values, including approximations of such given values resulting from experimental and/or measurement conditions, as reasonably deduced by one of ordinary skill in the art.

To provide a more concise description, some quantitative expressions herein are recited as a range from about X to about Y. It should be understood that when a range is recited, the range is not limited to the recited upper and lower limits, but rather, includes the entire range of about X to about Y amounts or any amount therebetween.

The eugenol D described herein can optionally be in the form of a finished package. In one embodiment, the package is a container such as a plastic, metal or glass tube or jar containing the eugenol D. The product may additionally have a package such as a plastic or cardboard box for storing the container. In one embodiment, the product comprises eugenol D and has instructions directing the user to apply eugenol D to the skin to treat signs of skin aging as discussed below. Such instructions may be printed on the container, on the label insert, or on any other package.

As used herein, "topical application" means directly applying or spreading on the external skin, scalp or hair, for example, using the hand or an applicator such as a wipe, roller or sprayer.

As used herein, "cosmetically acceptable" means that the ingredients described by the term are suitable for use in contact with tissue (e.g., skin or hair) without undue toxicity, incompatibility, instability, irritation, allergic response, or the like.

The present invention finds use in treating signs of skin aging with eugenol D. As used herein, "signs of skin aging" include the presence of fine lines and wrinkles, loss of elasticity, uneven skin tone, and scarring. In a particularly preferred embodiment, the sign of aging is the presence of fine lines and wrinkles and/or loss of elasticity.

As used herein, "treating signs of skin aging" refers to reducing, preventing, ameliorating, or eliminating the presence or signs of skin aging described above.

As used herein, "wrinkles" includes fine lines, fine wrinkles, or coarse wrinkles. Examples of wrinkles include, but are not limited to, fine lines around the eyes (e.g., "fish tail lines"), forehead and cheek wrinkles, eyebrow lines, and smiles around the mouth.

As used herein, "loss of elasticity" includes loss of elasticity or structural integrity of skin or tissue, including but not limited to sagging, laxity, and loose tissue. Loss of elasticity or tissue structural integrity may be caused by a variety of factors including, but not limited to, disease, aging, hormonal changes, mechanical trauma, environmental damage, or as a result of the application of a product such as a cosmetic or pharmaceutical to the tissue.

As used herein, "uneven skin tone" refers to skin conditions associated with diffuse or mottled pigmentation, which may be classified as hyperpigmentation, such as post-inflammatory hyperpigmentation.

As used herein, "scar" means a skin condition associated with redness or erythema.

As used herein, "cosmetic" refers to a cosmetic substance or article that retains, restores, imparts, stimulates or enhances the appearance of a body or appears to enhance a beauty or youthful appearance, particularly when it relates to the appearance of tissue or skin.

As used herein, "cosmetically effective amount" means an amount of physiologically active compound or composition sufficient to treat one or more signs of skin aging, but low enough to avoid serious side effects. The cosmetically effective amount of a compound or composition will vary depending on: the particular 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 the other treatments, the particular compound or product/composition employed, the particular cosmetically acceptable carrier utilized, and the like.

Cat's eye herb phenol D

The present invention is based on the following unexpected findings: the opal D has effects of promoting fibroblast to generate collagen and elastin. Therefore, the euonymus alatus phenol D can be used as an effective component to be added into skin care products to help improve skin relaxation, wrinkle problems and the like. The invention aims to provide the efficacy of the opal D for promoting the generation of collagen and elastin and the application of the opal D in cosmetics.

In the use of the agent to promote collagen and/or elastin production by fibroblasts, the concentration of equol D is at least 0.08. Mu.g/mL, preferably at least 0.1. Mu.g/mL.

In some embodiments, the concentration of equol D used in applications that promote collagen and/or elastin production by fibroblasts is preferably less than 0.225 μg/mL. In some embodiments, the working concentration of the eugenol D is 0.001-0.225 μg/mL, preferably 0.01-0.225 μg/mL, more preferably 0.1-0.225 μg/mL.

External preparation for skin

In some embodiments, the eugenol D can be used in the preparation of a skin external agent. 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 content of the eugenol D in the skin external preparation may be 0.000001 to 1% by weight, preferably 0.00001 to 1% by weight, more preferably 0.001 to 1% by weight, and still more preferably 0.01 to 1% by weight.

In some embodiments, the content of eugenol D in the skin external agent is 0.000008 to 20 wt%, preferably 0.00001 to 10 wt%.

The external skin preparation is a general concept of all ingredients commonly 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 skin external preparation comprising the eugenol D 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 the opal D can be prepared 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.

Although it is preferred that the skin external preparation comprising the eugenol D comprises water, the skin external preparation may alternatively be anhydrous or an ointment that does not comprise water but rather is an organic and/or silicone solvent, grease, lipid, and wax. 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 comprising the eugenol D 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 skin external preparation comprising the opal D 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 preparation 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, etc., as long as they are physically and chemically compatible with the other components of the skin external preparation and do not affect the effects of the equol D 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.

Additional cosmetic active agents

In some embodiments, the skin external preparation may further comprise additional cosmetic active agents. As used herein, a "cosmetically active agent" is a compound that has a cosmetic or therapeutic effect on skin or hair (e.g., a synthetic compound or a compound isolated from a natural source or natural extract), including but not limited to anti-acne agents, oil control agents, antimicrobial agents, anti-inflammatory agents, antifungal agents, antiparasitic agents, topical analgesics, sunscreens, photoprotective agents, antioxidants, keratolytic agents, surfactants, moisturizers, nutrients, vitamins, energy enhancers, antiperspirants, astringents, deodorants, solidifying agents, anti-sclerokeratotic agents, and agents for hair and/or skin conditioning.

In one embodiment, these cosmetically active agents are selected from (but are not limited to): hydroxy acids, benzoyl peroxide, D-panthenol, octyl methoxycinnamate, titanium dioxide, octyl salicylate, homosalate, avobenzone, carotenoids, 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 estrogens, 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 vera, feverfew, oatmeal, and derivatives and mixtures thereof. The cosmetically active agent is typically 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 skin external agent of the present invention.

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., alpha, beta, gamma, or delta tocopherol) or mixtures thereof, and derivatives thereof.

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

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

Application method

The skin external preparation of the present invention can be topically applied to mammalian skin in need of treatment for one or more signs of skin aging as described above. In one embodiment, the skin external agent may be applied to skin in need of treatment for fine lines and wrinkles and/or loss of elasticity. The skin external agent may be applied to the skin in need of such treatment according to a suitable treatment regimen, such as monthly, weekly, every other day, daily, twice daily, etc.

In certain embodiments, the skin external preparations of the present invention may also be used to address other needs associated with the skin. For example, the skin external preparation of the present invention can be used for treating post-inflammatory hyperpigmentation, for reducing pore size, for reducing sebum production, and for alleviating scars. In certain other embodiments, the skin external agents of the present invention may be applied simultaneously with or within hours of a mechanical or physical exfoliating treatment (e.g., microdermabrasion treatment), or simultaneously with a chemical exfoliating agent or a keratolytic agent such as salicylic acid. In certain other embodiments, the skin external agents of the present invention may be applied to mucous membranes or other tissues such as vaginal tissue, oral tissue, or ocular tissue. In certain other embodiments, the skin external preparations of the present invention may be applied to mild wounds or post-operative sites to promote healing, to insect bites, to poison vine skin disorders or similar skin conditions, or are generally used to reduce itching.

Examples

The invention will be further illustrated by the following examples. It is noted herein that the examples are given solely for the purpose of illustration and are not to be construed as limitations on the scope of the invention, since many insubstantial modifications and variations will become apparent to those skilled in the art in light of the above teachings. The test methods in the following 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.

The purity of the opal D used in the invention is more than 90%, and the opal D is derived from sweet wormwood and provided by Chinese medical institute of traditional Chinese medicine.

Example 1: preparation of euonymus Alata phenol D

3.6mg of eugenol D was weighed, and the volume was fixed with dimethylsulfoxide (Sigma, DMSO) to a 10mL volumetric flask to obtain 0.36mg/mL of eugenol D solution for use.

Example 2: preparation of euonymus Alata phenol D

The eugenol D1.8mg is weighed, and the volume is fixed to a 10mL volumetric flask by using DMSO to obtain a 0.18mg/mL eugenol D solution for later use.

Example 3: preparation of euonymus Alata phenol D

The method comprises the steps of weighing 9.0mg of the eugenol D, and fixing the volume to a 100mL volumetric flask by using DMSO to obtain 0.09mg/mL of eugenol D solution for later use.

Example 4 preparation of Tikola phenol D

The eugenol D10.8mg is weighed, and the volume is fixed to a 10mL volumetric flask by using DMSO to obtain 1.08mg/mL eugenol D solution for later use.

Test example 1: fibroblast cytotoxicity test

Cell inoculation: skin fibroblast (Fb 20081902, guangdong Boxi Biotechnology Co., ltd.) was cultured at a rate of 8X 10 ³ Seed Density of each well cells were seeded into 96 well plates, incubator (Thermo, 150I) (37 ℃, 5% CO ₂ ) Incubate overnight.

Test grouping: the test set-up was zeroed, solvent control, positive control and opal D.

Example 18 concentration gradients were set, with 3 duplicate wells set per concentration gradient.

Preparing liquid: the solutions were prepared as follows.

TABLE 1

Administration: and when the cell plating rate in the 96-well plate reaches 40% -60%, the administration is carried out. 200. Mu.L of DMEM medium (Guangdong Boxi Biotechnology Co., ltd.) was added to each well of the solvent control group; 200. Mu.L of culture solution containing 10% DMSO is added to each well of the positive control group; example groups 200. Mu.L of the culture medium containing the examples at the corresponding concentrations was added per well; the zeroed group was inoculated without cells and only 200. Mu.L of cell culture medium was added. After the completion of the administration, the 96-well plate was placed in an incubator for culturing for 24 hours.

And (3) detection: after incubation of the cells for 24 hours, the supernatant was discarded, 0.5mg/mL MTT (Sigma) was added, incubated at 37℃for 4 hours in the absence of light, after the incubation was completed, 150. Mu. LDMSO was added to each well, OD values were read at 490nm and cell viability was calculated. Results are expressed as mean±sd.

The toxicity judging method comprises the following steps: the OD value of the sample group after MTT detection is compared with the OD value of the control group to judge, and after turning points are screened, 2 concentrations are respectively selected to conduct morphological photographing on the upper and lower positions of the turning points, so that the MTT result is verified.

Experimental results:

TABLE 2

From the MTT and morphological results (Table 2 and FIGS. 1-6), FIG. 2 shows that the fibroblast morphology is slightly contracted, the cell density is significantly reduced, and the cytoplasmic transparency is reduced; FIG. 3 shows that the fibroblast is cleared from the edge, the morphology is still good, the cell density is slightly reduced, and the difference between FIGS. 2-3 and 1 is obvious. FIGS. 4-6 show clear edges of fibroblasts, expanded morphology, greater cell density, homogeneous and transparent cells, and no significant difference from FIG. 1 (solvent control). It was shown that opal D showed no significant cytotoxicity at a mass concentration of 0.225 μg/mL based on fibroblasts.

Test example 2: fibroblast-based Collagen I content test

Cell inoculation: according to 4X 10 ⁴ Cell/well seeding density cells were seeded into 24-well plates and incubated overnight in incubator (37 ℃, 5% co 2).

Preparing liquid: the group was divided into a blank group, a negative control group, a TGF-. Beta.1 group (0.1. Mu.g/mL) and a group of example 1 (0.225. Mu.g/mL and 0.1125. Mu.g/mL).

Administration: and (3) when the cell plating rate in the 24-well plate reaches 40% -60%, administration is carried out. 2mL of sample is added to each well, and 3 compound wells are arranged in each group. After the administration, the 24-well plate was placed in an incubator to be cultured for 24 hours.

UVA irradiation: the groups requiring UVA irradiation were subjected to UVA irradiation of 30J/cm2 and placed in an incubator for continuous cultivation for 24 hours.

And (3) sample collection: after 24 hours, the cell culture supernatant was collected in an EP tube and stored in a freezer at-80 ℃.

ELISA detection: the detection was performed according to the instructions of the Collagen I ELISA kit (Human Collagen Type I (Col-I), CUSABIO Co.). The Collagen I content results are expressed as mean.+ -. SD.

Experimental results of type I Collagen (Collagen I):

TABLE 3 Table 3

Compared with the blank control group, the Collagen I content of the negative control group is reduced from 26.99ng/mL to 22.13ng/mL, which indicates that the test stimulation condition is effective.

The increase in the Collagen I content of the positive control (TGF-. Beta.) from 22.13ng/mL to 31.44ng/mL compared to the negative control indicates that the positive control was effective. The content of the Collagen I is increased from 22.13ng/mL to 24.66ng/mL when the mass concentration of the Catalaxyl D is 0.1125 mug/mL, and is increased from 22.13ng/mL to 31.43ng/mL when the mass concentration of the Catalaxyl D is 0.225 mug/mL. The result shows that the opal D can well promote fibroblasts to generate Collagen I, and the anti-wrinkle effect is achieved.

Test example 3: fibroblast-based Elastin content assay

ELISA detection: detection was performed according to the instructions of Elastin ELISA kit (Human Elastin ELISA Kit, abcam). The Elastin content results are expressed as mean±sd.

Elastin (Elastin) experimental results:

TABLE 4 Table 4

Compared with the blank control group, the Elastin content of the negative control group is reduced from 522.08ng/mL to 265.78ng/mL, which indicates that the test stimulation condition is effective.

Compared with the negative control group, the Elastin content of the positive control group is increased from 265.78ng/mL to 890.37ng/mL, which proves that the positive control is effective. The content of Elastin is increased from 265.78ng/mL to 427.47ng/mL when the mass concentration of the opal D is 0.1125 mug/mL, and from 265.78ng/mL to 473.00ng/mL when the mass concentration of the opal D is 0.225 mug/mL. The result shows that the opal D can well promote the fibroblast to generate Elastin, thereby achieving the effect of tightening.

The euonymus alatus phenol D can be used for preparing medicines, health-care foods or 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 content of the eugenol D in the medicine, the health food or the skin external preparation may be 0.000008 to 10% by weight, preferably 0.00001125 to 5% by weight, more preferably 0.000015 to 5% by weight, and still more preferably 0.0000225 to 1% by weight.

The following are examples of specific applications of the euonymus alatus phenol D in medicines, health foods or external skin preparations, and formulations and preparation methods of the formulations. In the following tables, "-" indicates no addition.

Application example 1: preparation of face cream

/>

Application example 2: preparation of the emulsion

Application example 3: preparation of jelly

Application example 4: preparation of toning lotion

Application example 5: preparation of essence

Application example 6: preparation of facial mask

Application example 7: preparation of eye cream

/>

Application example 8: preparation of aerosol (cleaning foam)

Application example 9: preparation of the spray

Application example 10: preparation of bath lotion

Example 11: preparation of facial cleanser

/>

Claims

1. Use of eugenol D to promote collagen and/or elastin production.

2. The use of claim 1, wherein the concentration of eugenol D is at least 0.08 μg/mL.

3. The use of claim 2, wherein the concentration of eugenol D is at least 0.1 μg/mL.

4. The use according to any one of claims 1 to 3, wherein the collagen is type I collagen.

5. The application of the euonymus alatus phenol D in preparing external skin preparations, medicines and health-care foods with the function of promoting the generation of collagen and/or elastin.

6. The use according to claim 5, wherein the collagen is type I collagen.

7. The use according to claim 5, wherein the content of the eugenol D in the external skin preparation, the pharmaceutical product and the health food is 0.000008 to 20 wt%.

8. The use according to claim 5, wherein the content of the eugenol D in the external skin preparation, the pharmaceutical product and the health food is 0.00001 to 10% by weight.

9. The use according to any one of claims 5 to 8, wherein the external skin preparation 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.