Application of osmundone in promoting generation of collagen and / or elastin

Abstract

The invention provides application of osmundone in promoting fibroblast proliferation and promoting generation of collagen and / or elastin. The invention also relates to application of osmundone in preparation of skin external preparations, medicines and health-care foods, which have the effects of promoting fibroblast proliferation and promoting collagen and / or elastin generation.

Description

Technical Field

[0001] The present invention relates to the field of natural drug chemistry, and in particular to the application of porphyrin to promote the production of collagen and / or elastin, and the application of porphyrin to prepare skin external preparations, medicines and health foods having the effect of promoting the production of collagen and / or elastin. Background Art

[0002] The determining factor of skin aging lies in the damage of collagen in the dermis by the internal and external environment. Collagen accounts for 72% of the skin and 80% of the dermis. Its three-dimensional spiral structure is like a dam in a reservoir, locking the moisture deep in the skin. It supports the skin like a spring. The human body can produce a lot of collagen when it is young, but its production will decrease with age. Elastin is the main component of elastic fiber. It allows the skin to stretch and fold, and is responsible for maintaining and supporting the elasticity of the skin. As we age, the elastin content in the skin decreases, thereby reducing the network support needed to maintain firm skin, and the skin begins to sag, fine lines and other aging phenomena.

[0003] Fibroblasts (Fb) are the main cells that make up the dermis of the skin and are the main cells that produce collagen and other intercellular matrix in the dermis. Their normal differentiation and proliferation maintain the normal structure and physiological function of the skin. Fb has a strong ability to synthesize proteins, and can synthesize and secrete a large number of matrix components such as elastin, collagen, glycosaminoglycans and glycoproteins, thereby generating elastic fibers, collagen fibers and reticular fibers, and secreting a variety of cell repair factors, which give the skin a strong renewal and self-repair ability, thereby increasing the thickness and density of the dermis of the skin, which has the potential to stretch wrinkles and restore skin elasticity and luster.

[0004] Osmundacetone, Chinese name 3,4-dihydroxybenzylideneacetone, English name Osmundacetone, Cas number 123694-03-1 or 37079-84-8, molecular formula C 10 H 10 O3, exists in Osmanthus fragrans and Inonotus obliquus. The structural formula of Osmanthus fragrans is:

[0005]

[0006] Modern synthesis routes basically use 3,4-dihydroxybenzaldehyde or 3,4-bis(methoxymethoxy)benzaldehyde as raw materials. Modern pharmacology shows that purpurogenol can regulate blood pressure and lower cholesterol.

[0007] For example, Chinese patent application 201780074399.X discloses a composition containing osfera ketone or a pharmaceutically acceptable salt thereof for preventing or treating bone diseases, and reports that Qi Zitong controls oil to improve symptoms of bone diseases and activates osteoblast differentiation. The patent also mentions that "the preferred content of the food composition of the present invention is 0.01-50wt%". At the same time, the patent application also shows that in all tests of human toxicity, irritation, carcinogenicity, environmental toxicity, etc. by many U.S. toxic substance regulatory agencies, purpurogenol was determined to be harmless and safe (Note: TSCA: not listed; CLEAN WATER ACT (CWA): not listed; SARA313: not listed; MARINE POLLUTANT: not listed; RIGHT TO KNOW LIST (NEW JERSEY): not listed; RIGHT TO KNOW LIST (MASSACHUSETTS): not listed; RIGHT TO KNOW LIST (PENNSYLVANIA): not listed; ILLINOISTOXIC AIR CONTAMINANTS: not listed; CLEAN AIR ACT (CAA): not listed; DHS CHEMICALS OF INTEREST: not listed; CALIFORNIA PROPOSITION 65: not listed). PROP65): Not listed; OSHA: Not listed; CALIFORNIAPROP 65TOXICITY TYPE(CANCER, DEVELOPMENTAL, FEMALE, MALE): None; OSHA HAZARD CLASS(CARCINOGEN, CORROSIVE, FLAMABLE, REACTIVE, TOXIC): None, etc.).

[0008] At present, there is no related report on the application of porphyrin in cosmetics. The present invention unexpectedly found that porphyrin has excellent effects of promoting Fb proliferation, collagen and elastin, and can be used as an effective additive in skin external preparations, medicines and health foods. Summary of the invention

[0009] The invention provides the application of porphyrin to promote the proliferation of fibroblasts and the production of collagen and / or elastin.

[0010] In a preferred embodiment, the collagen is type I collagen.

[0011] In a preferred embodiment, porphyrin is used at a concentration of at least 0.01 μg / mL, more preferably at least 0.05 μg / mL.

[0012] The present invention also relates to the use of porphyrin in the preparation of external skin preparations, medicines and health foods that have the functions of promoting the proliferation of fibroblasts and the production of collagen and / or elastin.

[0013] In a preferred embodiment, the content of porphyrin in the external skin preparation, medicine and health food is 0.001-20% by weight, more preferably 0.005-10% by weight, more preferably 0.01-5% 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.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The proliferation effect of porphyrin on fibroblasts was shown.

[0017] Figure 2 Cell morphology of solvent control toxicity to fibroblasts is shown. Microscope Olympos CKX53, camera Canon EOS 750D, magnification 200x.

[0018] Figure 3 The cell morphology of 0.25% Example 1 toxicity to fibroblasts is shown. Microscope: OlymposCKX53, camera: Canon EOS 750D, magnification: 200 times.

[0019] Figure 4 The cell morphology of 0.125% Example 1 toxicity to fibroblasts is shown. Microscope: Olympos CKX53, camera: Canon EOS 750D, magnification: 200 times.

[0020] Figure 5 The cell morphology of 0.0625% Example 1 toxicity to fibroblasts is shown. Microscope: OlymposCKX53, camera: Canon EOS 750D, magnification: 200 times.

[0021] Figure 6 The cell morphology of 0.0313% Example 1 toxicity to fibroblasts is shown. Microscope: OlymposCKX53, camera: Canon EOS 750D, magnification: 200 times.

[0022] Figure 7The cell morphology of 0.0078% Example 1 toxicity to fibroblasts is shown. Microscope: OlymposCKX53, camera: Canon EOS 750D, magnification: 200 times.

[0023] Figure 8 Collagen I content results are shown.

[0024] Fig. 9 Elastin content results are shown. DETAILED DESCRIPTION

[0025] The present invention relates to the effect of porphyrin on promoting the proliferation of fibroblasts and the production of collagen and / or elastin. It is discovered for the first time that porphyrin can be used as a functional additive and applied to external skin preparations, medicines and health foods to help achieve the effects of maintaining skin firmness, improving skin sagging and / or wrinkles, and resisting skin aging.

[0026] 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.

[0027] 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.

[0028] Osirhinone described herein can be optionally in the form of finished product packaging. In one embodiment, the packaging is a container such as plastic, metal or glass tube or wide-mouth bottle that osirhinone is housed. The product can additionally have a packaging such as plastic or cardboard box for storing the container. In one embodiment, the product comprises osirhinone and has instructions for guiding the user to apply osirhinone to the skin to treat skin aging signs as discussed below. This instruction can be printed on the container, label insert or any other packaging.

[0029] 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.

[0030] As used herein, "cosmetically acceptable" means that the ingredients described by the term are suitable for use in contact with tissue (eg, skin or hair) without undue toxicity, incompatibility, instability, irritation, allergic response, or the like.

[0031] The present invention finds that porphyrin is useful for treating signs of skin aging. 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 signs of aging are the presence of fine lines and wrinkles and / or loss of elasticity.

[0032] As used herein, "treating the signs of skin aging" means alleviating, reducing, preventing, ameliorating or eliminating the presence or signs of skin aging as described above.

[0033] As used herein, "wrinkles" include fine lines, micro wrinkles or macro wrinkles. Examples of wrinkles include, but are not limited to, fine lines around the eyes (e.g., "crow's feet"), forehead and cheek wrinkles, frown lines, and smile lines around the mouth.

[0034] As used herein, "loss of elasticity" includes loss of elasticity or structural integrity of skin or tissue, including but not limited to sagging, loose and loose tissue. Loss of elasticity or tissue structural integrity can 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 applying products such as cosmetics or drugs to the tissue.

[0035] As used herein, "uneven skin tone" refers to a skin condition associated with diffuse or mottled pigmentation that can be classified as hyperpigmentation, such as post-inflammatory hyperpigmentation.

[0036] As used herein, "scarring" means a skin condition associated with redness or erythema.

[0037] 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.

[0038] As used herein, "cosmetically effective amount" means an amount of a 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 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.

[0039] Porphyrone

[0040] The present invention is based on the following unexpected discovery: osfera ketone has the effect of promoting fibroblast proliferation and promoting human fibroblasts to produce collagen and / or elastin. Therefore, osfera ketone 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 osfera ketone in promoting fibroblast proliferation and promoting human collagen and / or elastin production and its application in cosmetics.

[0041] In the application of promoting fibroblast proliferation, the concentration of porphyrin is at least 0.1 μg / mL, more preferably at least 1 μg / mL.

[0042] In the application of promoting fibroblasts to produce collagen and / or elastin, the concentration of porphyrin is at least 0.01 μg / mL, more preferably at least 0.05 μg / mL.

[0043] In some embodiments, the concentration of porphyrone used is preferably less than 20 μg / mL, more preferably less than 15 μg / mL, more preferably less than 1 μg / mL, and more preferably less than 0.1 μg / mL.

[0044] In some embodiments, porphyrin is used at a concentration of 0.01-1 μg / mL, preferably 0.05-1 μg / mL, and more preferably 0.05-0.1 μg / mL.

[0045] Skin topical products

[0046] In some embodiments, porphyrin can be used for the preparation of a skin topical preparation. The skin topical preparation is preferably a cosmetic composition, including but not limited to products in the form of cream, lotion, gel, toner, essence, mask, eye cream, aerosol (cleansing foam), spray, shower gel and facial cleanser.

[0047] The content of porphyrin in the external skin preparation is 0.001-20% by weight, preferably 0.005-10% by weight, and more preferably 0.01-5% by weight.

[0048] 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.

[0049] The skin topical preparation comprising porphyrone can be topically applied to human skin and / or hair. The skin topical preparation can also include a cosmetically acceptable topical carrier, which can be about 50% by weight to about 99.99% by weight of the skin topical preparation (such as about 80% by weight to about 99% by weight of the skin topical 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.

[0050] 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.

[0051] The topical skin preparation containing porphyrin 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] Although it is preferred that the topical skin preparation containing porphyrin contains water, alternatively the topical skin preparation may be anhydrous or an ointment containing no water but organic and / or silicone solvents, greases, lipids and waxes. The ointment may contain a simple base of animal or vegetable oils or semisolid hydrocarbons. The ointment 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] The skin topical preparation comprising osfera ketone can also be formulated into a gel (e.g., an aqueous gel, an alcoholic gel, an alcohol / water-type gel or an oily gel using a suitable gelling agent). Suitable gelling agents for aqueous gels 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 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% and 5% by weight of such gelling agents.

[0060] The external skin preparation containing porphyrin may also be formulated into a solid preparation (eg, a wax-based stick, a bar soap, a powder, or a wipe containing the powder).

[0061] 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.

[0062] The skin topical preparation of the present invention may contain additional components commonly found in skin care compositions, such as emollients, skin regulators, emulsifiers, preservatives, antioxidants, fragrances, chelating agents, etc., as long as they are physically and chemically compatible with other components in the skin topical preparation and do not affect the effects of the porphyrin of the present invention.

[0063] 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 %.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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 %.

[0069] 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.

[0070] Additional cosmetic active agents

[0071] 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.

[0072] 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.

[0073] 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.

[0074] Examples of hydroxy acids include, but are not limited to, glycolic acid, lactic acid, malic acid, salicylic acid, citric acid, and tartaric acid.

[0075] 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.

[0076] How to use

[0077] The skin topical of the present invention can be topically applied to mammalian skin in need of treating one or more signs of skin aging as described above. In one embodiment, the skin topical can be applied to skin in need of treating fine lines and wrinkles and / or loss of elasticity. The skin topical can be applied to skin in need of such treatment according to a suitable treatment regimen, such as monthly, weekly, every other day, daily, twice a day, etc.

[0078] In certain embodiments, the skin topical preparations of the present invention can also be used to treat other needs related to the skin. For example, the skin topical preparations of the present invention can be used to treat hyperpigmentation after inflammation, to reduce pore size, to reduce sebum production, and to alleviate scars. In certain other embodiments, the skin topical preparations of the present invention can be applied simultaneously with a mechanical or physical epidermal exfoliation treatment (e.g., microdermabrasion treatment) or within a few hours of the treatment, or simultaneously with a chemical peel or keratolytic agent such as salicylic acid. In certain other embodiments, the skin topical preparations of the present invention can be applied to mucous membranes or other tissues such as vaginal tissue, oral tissue, or eye tissue. In certain other embodiments, the skin topical preparations of the present invention can be applied to mild wounds or postoperative sites to promote healing, to insect bites, to poison ivy skin disorders or similar skin conditions, or generally used to relieve itching.

[0079] Example

[0080] 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.

[0081] The porphyrin used in the examples of the present invention was provided by the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences.

[0082] Example 1: Preparation of porphyrone

[0083] Weigh 1.78 mg of porphyrin ketone and dilute it to a 10 mL volumetric flask with dimethyl sulfoxide (Sigma, DMSO) to obtain a 0.178 mg / mL porphyrin ketone solution for later use.

[0084] Example 2: Preparation of porphyrone

[0085] Weigh 8.9 mg of porphyrin ketone and dilute to 10 mL volumetric flask with DMSO to obtain 0.89 mg / mL porphyrin ketone solution for later use.

[0086] Example 3: Preparation of porphyrone

[0087] Weigh 5.34 mg of porphyrin ketone and dilute to 10 mL volumetric flask with DMSO to obtain 0.534 mg / mL porphyrin ketone solution for later use.

[0088] Example 4: Preparation of porphyrone

[0089] Weigh 17.8 mg of porphyrin ketone and dilute to 10 mL volumetric flask with DMSO to obtain a 1.78 mg / mL porphyrin ketone solution for later use.

[0090] Example 5: Evaluation of the effect of porphyrin on the proliferation of fibroblasts

[0091] The CCK-8 method is a highly sensitive, non-radioactive colorimetric assay used to determine the number of live cells in cell proliferation or toxicity tests. The reagent contains 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt (WST-8), which, under the action of electron carriers, can reduce the dehydrogenase in the cell mitochondria to a highly water-soluble yellow formazan product. The amount of formazan generated is positively correlated with the degree of cell proliferation. The CCK-8 method can calculate the number of live cells through colorimetric determination, and measure its optical density with an enzyme-linked immunosorbent assay. The proliferation rate of the test group can be obtained by comparing it with the control group, thereby determining the cytotoxicity or proliferation-promoting activity of the test sample.

[0092] 1 Experimental methods:

[0093] (1) Preparation of 2× sample solutions: Example 2 was prepared using DMEM medium (Gibco) into 24.48 μg / mL, 14.24 μg / mL, 7.12 μg / mL, 3.56 μg / mL, and 1.78 μg / mL solutions for later use.

[0094] (2) Cell inoculation: Fibroblasts (Guangdong Boxi Biotechnology Co., Ltd.) were seeded at 7.5×10 3 The cells were seeded into a 96-well plate at a seeding density of 100 cells / well and incubated in an incubator (Thermo, HERACELL240i) (37° C., 5% CO 2 ) for 24 hours.

[0095] (3) Experimental Grouping: The experiment set up a zero-adjustment group, a negative control group, a positive control group and Example 2.

[0096] In Example 2, five concentration gradients were set, and four replicate wells were set under each concentration gradient.

[0097] (4) Administration: 200 μL of DMEM culture medium was added to each well of the negative control group; 200 μL of 0.04 μg / mL epidermal growth factor (EGF) was added to each well of the positive control group; 200 μL of culture medium containing the corresponding concentration of the example was added to each well of the example group; the zero adjustment group was not inoculated with cells, and only 200 μL of cell culture medium was added. After administration, the 96-well plate was placed in an incubator and cultured for 48 hours.

[0098] (5) CCK-8 assay: According to the number of wells required for assay, add 0.01 mL of CCK-8 working solution to 0.1 mL of DMEM medium without fetal calf serum (FCS) per well. Take out the 96-well plate to be assayed, discard the medium, add 0.1 mL of CCK-8 working solution to each well, read the OD value at 450 nm and calculate the cell viability.

[0099]

[0100] (6) Experimental results:

[0101] Table 1

[0102]

[0103] Compared with the negative control group, the cell viability of the positive control group was significantly improved, indicating that the positive control of this test was effective; when the mass concentration of Example 2 was 1.78 μg / mL to 14.24 μg / mL, the cell viability was significantly improved, achieving the goal of promoting fibroblast proliferation.

[0104] Example 6: Evaluation of the performance of porphyrone on collagen and elastin

[0105] 1. Toxicity test experimental method

[0106] Cell inoculation: Skin fibroblasts (Guangdong Boxi Biotechnology Co., Ltd., Fb20081902) were plated at 8×10 3 The cells were seeded into a 96-well plate at a seeding density of 100 cells / well and incubated overnight in an incubator (Thermo, 150I) (37°C, 5% CO2).

[0107] Experimental groups: The experiment set up a zero-adjustment group, a solvent control group, a positive control group and Example 1.

[0108] In Example 1, 8 concentration gradients were set, and 3 replicate wells were set under each concentration gradient.

[0109] Solution preparation: Prepare the solution according to the table below.

[0110] Table 2

[0111]

[0112] Administration: Administration was performed when the cell plating rate in the 96-well plate reached 40% to 60%. 200 μL of DMEM culture medium (Guangdong Boxi Biotechnology Co., Ltd.) was added to each well of the solvent control group; 200 μL of culture medium containing 10% DMSO was added to each well of the positive control group; 200 μL of culture medium containing the corresponding concentration of the embodiment was added to each well of the embodiment group; the zeroing group had no cell inoculation and only 200 μL of cell culture medium was added. After administration, the 96-well plate was placed in an incubator for 24 hours.

[0113] Detection: After incubating the cells for 24 hours, the supernatant was discarded, 0.5 mg / mL MTT (Sigma) was added, and the cells were incubated at 37°C in the dark for 4 hours. After the incubation, the supernatant was discarded, 150 μL DMSO was added to each well, the OD value was read at 490 nm, and the cell survival rate was calculated.

[0114]

[0115] Experimental results:

[0116] Table 3

[0117]

[0118] According to the MTT and morphological results (Table 3 and Figure 2-7 ) It can be seen that Figure 3 The fibroblasts showed slight shrinkage in morphology, with a significant decrease in cell density and cytoplasmic transparency; Figure 4 The fibroblasts have clear edges and the cell density is slightly decreased. Figure 3-4 and Figure 2 The difference is obvious. Figure 5-7 Fibroblasts have clear edges, stretched morphology, high cell density, homogeneous and transparent cells. Figure 2 (Solvent control group) had no significant difference. It showed that porphyrin ketone did not show obvious cytotoxicity based on fibroblasts within a mass concentration of 0.11125 μg / mL.

[0119] 2. Experimental methods for testing firming and anti-wrinkle effects

[0120] Cell seeding: 4×10 4 The cells were seeded into a 24-well plate at a seeding density of 100 cells / well and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0121] Solution preparation: divided into blank control group, negative control group, TGF-β1 group (0.1 μg / mL) and Example 1 group (0.11125 μg / mL and 0.055625 μg / mL).

[0122] Dosing: Dosing is performed when the cell plating rate in the 24-well plate reaches 40% to 60%. 2 mL of sample is added to each well, and 3 replicate wells are set up for each group. After dosing, the 24-well plate is placed in an incubator for 24 hours.

[0123] UVA irradiation: 30J / cm for the group that needs UVA irradiation 2 The cells were then placed in an incubator and exposed to UVA for 24 hours.

[0124] Sample collection: After 24 hours, collect the cell culture supernatant into an EP tube and store it in a -80°C refrigerator.

[0125] ELISA detection: The detection was performed according to the operating instructions of CollagenⅠELISA Kit (Human Collagen Type I (Col-I), CUSABIO) and Elastin ELISA Kit (Human Elastin ELISA Kit, Abcam).

[0126] Results Statistical analysis: The results were expressed as Mean ± SD. The t-test was used for statistical analysis for comparison among the groups. P < 0.05 was considered to be significantly different, and P < 0.01 was considered to be extremely significantly different.

[0127] CollagenⅠexperimental results:

[0128] Table 4

[0129]

[0130]

[0131] Note: When using the t-test method for statistical analysis, the significance compared with the blank control group is indicated by #; the significance compared with the negative control group is indicated by *.

[0132] Compared with the blank control group, the content of Collagen I in the negative control group decreased from 26.99 ng / mL to 22.13 ng / mL, indicating that the stimulation conditions of this test were effective.

[0133] Compared with the negative control group, the content of Collagen I in the positive control group (TGF-β1) increased from 22.13ng / mL to 31.44ng / mL, indicating that the positive control in this test is effective. When the mass concentration of porphyrin is 0.055625μg / mL, the content of Collagen I increased from 22.13ng / mL to 26.07ng / mL, indicating that porphyrin can well promote fibroblasts to produce Collagen I and achieve anti-wrinkle effect.

[0134] Elastin experimental results:

[0135] Table 5

[0136]

[0137] Compared with the blank control group, the Elastin content in the negative control group decreased from 522.08 ng / mL to 265.78 ng / mL, indicating that the stimulation conditions of this test were effective.

[0138] Compared with the negative control group, the Elastin content of the positive control group increased from 265.78ng / mL to 890.37ng / mL, indicating that the positive control of this test is effective. When the mass concentration of porphyrin was 0.055625μg / mL, the Elastin content increased from 265.78ng / mL to 343.22ng / mL; when the mass concentration was 0.11125μg / mL, the Elastin content increased from 265.78ng / mL to 303.91ng / mL, indicating that porphyrin can promote fibroblasts to produce Elastin and achieve a firming effect.

[0139] Osorbitolone can be used for the preparation of skin topical preparations. The skin topical preparation is preferably a cosmetic composition, such as a toner, an essence, a cream, etc. The weight percentage in the skin topical preparation is 0.0000055%-10% (w / w). The preferred weight percentage is 0.000011%-5% (w / w). The more preferred weight percentage is 0.000178%-1% (w / w). The most preferred weight percentage is 0.00142%-0.5% (w / w).

[0140] The following are examples of specific applications of porphyrin in external skin preparations, and the formulations and preparation methods of these preparations. In the following tables, "-" means no addition.

[0141] Application Example 1: Preparation of facial cream

[0142]

[0143]

[0144] Application Example 2: Preparation of Emulsion

[0145]

[0146] Application Example 3: Preparation of Gel

[0147]

[0148]

[0149] Application Example 4: Preparation of lotion

[0150]

[0151] Application Example 5: Preparation of Essence

[0152]

[0153] Application Example 6: Preparation of facial mask

[0154]

[0155]

[0156] Application Example 7: Preparation of Eye Cream

[0157]

[0158] Application Example 8: Preparation of Aerosol (Cleaning Bubble)

[0159]

[0160] Application Example 9: Preparation of Spray

[0161]

[0162] Application Example 10: Preparation of shower gel

[0163]

[0164]

[0165] Application Example 11: Preparation of facial cleanser

[0166]

[0167]

Claims

1. Application of porphyrin to promote fibroblast proliferation and promote collagen and / or elastin production.

2. The use according to claim 1, characterized in that The collagen is type I collagen.

3. The use according to claim 1, characterized in that The concentration of porphyrone used is at least 0.01 μg / mL.

4. The use according to claim 3, characterized in that The concentration of porphyrone used is at least 0.05 μg / mL.

5. The use of porphyrin in the preparation of skin external preparations, medicines and health foods that have the effects of promoting fibroblast proliferation and promoting collagen and / or elastin production.

6. The use according to claim 5, characterized in that The collagen is type I collagen.

7. The use according to claim 5, characterized in that The content of porphyrin in skin external preparations, medicines and health foods is 0.001-20% by weight.

8. The use according to claim 7, characterized in that The content of porphyrin in skin external preparations, medicines and health foods is 0.005-10% by weight.

9. The use according to claim 7, characterized in that The content of porphyrin in skin external preparations, medicines and health foods is 0.01-5% by weight.

10. 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.

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