Anti-inflammatory soothing effect and application of osmundone

Abstract

The invention provides an anti-inflammatory soothing application of osmundone. The anti-inflammatory soothing can be realized by inhibiting the activity of hyaluronidase. Anti-inflammatory relief can also be achieved by inhibiting IL-6, TNF-alpha and / or PGE2. The invention also relates to application of osmundone in preparation of external skin agents, medicines and health-care foods with anti-inflammatory and soothing effects. The external skin agents are selected from face cream, emulsion, gel, toning lotion, essence, masks, eye cream, aerosol cleaning foam, spray, shower gel or facial cleanser.

Description

Technical Field

[0001] The invention relates to the field of natural drug chemistry, and in particular to the anti-inflammatory and soothing effects of porphyrin and its application in external skin preparations, medicines and health foods. Background Art

[0002] Skin irritation, also known as primary irritation, is one of the common adverse reactions in cosmetics. It refers to the skin reaction caused by the irritating ingredients in cosmetics through non-immune mechanisms. Different individuals have different sensitivities to irritating ingredients, but if the irritation is strong enough, anyone can cause skin irritation. There are three main types of skin irritation: one is slight skin damage, but there is no irritation that causes skin inflammation, which is called irritant reaction; the second is that irritants (such as strong acids, strong alkalis, etc.) cause skin irritation in high concentrations and in a short period of time, so that the skin quickly produces an inflammatory reaction, which is called acute irritant inflammation; the third is long-term repeated use of cosmetics containing mild irritating ingredients, which causes skin inflammation, which is called chronic irritant inflammation, and acute and chronic irritant dermatitis are called irritant contact dermatitis. Generally speaking, cosmetics do not contain highly irritating substances, so among the adverse reactions of cosmetics, skin irritation is generally manifested as irritant reaction and chronic irritant inflammatory reaction. Skin irritation is mainly manifested as subjective symptoms such as burning, stinging, and itching, accompanied by objective signs such as redness, dryness, desquamation and edema.

[0003] Skin irritation is a skin reaction caused by multiple factors. Depending on the different types and structures of irritants, the pathways leading to skin irritation may also be different. Although there are many pathways to induce skin irritation, irritants first damage the skin surface, resulting in impaired barrier function. When the barrier function is impaired, it will lead to increased transepidermal water loss, causing skin desquamation and erythema. This process is a mild skin injury that has not yet reached the level of skin inflammation. However, as the irritant continues to act, the skin barrier function is further damaged, and the irritant can further enter the skin, thereby interacting with the cells inside the skin, causing changes in cell structure. Irritants can destroy the cell membrane to release cytoplasm containing the pro-inflammatory factor IL-1α, and IL-1α will further induce the expression of inflammatory factors such as IL-6, IL-8, TNF-α, PGE2, etc., leading to the occurrence of skin irritation, and finally the typical symptoms of irritant contact dermatitis appear.

[0004] In addition, under normal circumstances, hyaluronic acid and hyaluronidase in the body are in a state of equilibrium. However, once this state of equilibrium is broken, it will cause the occurrence and development of many diseases. Modern pharmacological studies have shown that hyaluronic acid also participates in immune regulation and has anti-inflammatory activity. Hyaluronidase can specifically degrade hyaluronic acid to improve the permeability and penetration of tissues, cell membranes, blood vessels, etc. When the content of hyaluronidase in the body is too high or the activity is too strong, it will affect the body's immune ability, making it easier for viruses, bacteria, etc. to invade the body and cause the body to induce a series of inflammations.

[0005] At present, common inflammatory relievers that inhibit hyaluronidase activity or inhibit the release of inflammatory factors include dexamethasone, sodium cromolyn, baicalin, paeonol, glycyrrhizic acid and its derivatives, etc. They have good anti-inflammatory and soothing abilities, among which dexamethasone and sodium cromolyn are the main positive controls for inhibiting inflammatory factors or inhibiting hyaluronidase activity.

[0006] 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:

[0007]

[0008] 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. At present, after consulting the literature, it is found that the literature only points out that the ethyl acetate part and the n-butanol part of the purpurogenol are effective anti-inflammatory parts, and it is not clear that the purpurogenol component in the purpurogenol is an effective anti-inflammatory ingredient, and there is no report on its application in cosmetics.

[0009] The present invention unexpectedly discovered that porphyrin can significantly inhibit the activity of hyaluronidase, while reducing the contents of IL-6, TNF-α, and PGE2, and has anti-inflammatory and soothing effects. Summary of the invention

[0010] The invention relates to the anti-inflammatory and soothing application of porphyrin

[0011] In some embodiments, the anti-inflammatory relief is achieved by inhibiting the activity of hyaluronidase.

[0012] In some embodiments, the anti-inflammatory relief is achieved by inhibiting IL-6, TNF-α and / or PGE2.

[0013] In a preferred embodiment, the concentration of porphyrone is at least 0.1 μg / mL, preferably at least 0.2 μg / mL.

[0014] On the other hand, the present invention also relates to the use of porphyrin in the preparation of external skin preparations, medicines and health foods with anti-inflammatory and soothing effects.

[0015] In a preferred embodiment, the content of porphyrin in the external skin preparation, medicine and health food is 0.001-20% by weight, preferably 0.005-10% by weight.

[0016] 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.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The inhibitory effect of porphyrin on hyaluronidase was shown. DETAILED DESCRIPTION

[0019] The present invention finds that porphyrin has anti-inflammatory and soothing effects and can be added as an effective ingredient to skin external preparations, medicines and health foods to help improve skin redness, tingling, allergies and the like.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Porphyrone

[0026] The present invention is based on the following unexpected discovery: osfera ketone has anti-inflammatory and soothing effects. Therefore, osfera ketone can be added as an effective ingredient to skin topical preparations, medicines and health foods to help improve skin redness, tingling, allergies and the like.

[0027] In anti-inflammatory and soothing applications, the concentration of porphyrone is at least 0.1 μg / mL, more preferably at least 0.2 μg / mL.

[0028] In some embodiments, the concentration of porphyrone used is preferably less than 0.5 μg / mL, more preferably less than 0.4 μg / mL, and more preferably less than 0.3 μg / mL.

[0029] In some embodiments, osferazone is used at a concentration of 0.1 μg / mL to 17.8 μg / mL. In some embodiments, osferazone is used at a concentration of 0.2 μg / mL to 17.8 μg / mL.

[0030] In some embodiments, osferazone is used at a concentration of 2.225 μg / mL to 17.8 μg / mL. In some embodiments, osferazone is used at a concentration of 4.9 μg / mL to 17.8 μg / mL.

[0031] Skin topical products

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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).

[0047] 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.

[0048] 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.

[0049] 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 %.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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 %.

[0055] 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.

[0056] Additional cosmetic active agents

[0057] 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.

[0058] 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.

[0059] 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.

[0060] Examples of hydroxy acids include, but are not limited to, glycolic acid, lactic acid, malic acid, salicylic acid, citric acid, and tartaric acid.

[0061] 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.

[0062] How to use

[0063] 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.

[0064] 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.

[0065] Example

[0066] 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.

[0067] 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.

[0068] Example 1: Preparation of porphyrone

[0069] Weigh 8.9 mg of porphyrin ketone and dilute it to a 10 mL volumetric flask with dimethyl sulfoxide (Sigma, DMSO) to obtain a 0.89 mg / mL porphyrin ketone solution for later use.

[0070] Example 2: Preparation of porphyrone

[0071] Weigh 1.78 mg of porphyrin ketone and dilute to 10 mL volumetric flask with DMSO to obtain a 0.178 mg / mL porphyrin ketone solution for later use.

[0072] Example 3: Preparation of porphyrone

[0073] 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.

[0074] Example 4: Preparation of porphyrone

[0075] 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.

[0076] Example 5: Evaluation of the inhibitory effect of porphyrone on hyaluronidase

[0077] High molecular weight hyaluronic acid plays an important role in regulating scarless repair of wound healing and can significantly reduce inflammatory response. However, the degradation products of hyaluronic acid can increase the inflammatory response during wound healing. The degradation reaction of hyaluronic acid depends on the activity of hyaluronidase. The inhibition of hyaluronidase activity can be used as an indicator of the anti-inflammatory response of the substance. In an acidic environment, hyaluronidase can hydrolyze the substrate hyaluronic acid and convert it into N-acetylglucosamine. N-acetylglucosamine produces a color reaction with dimethylaminobenzaldehyde. The activity of hyaluronidase is determined by the depth of color.

[0078] 1 Experimental methods:

[0079] (1) Preparation of pH 3.5 buffer: Dissolve 1.3609 g of sodium acetate trihydrate (National Pharmaceutical Reagent, analytical grade) in 100 mL of water and stir evenly with a glass rod to prepare a 0.1 M solution A for later use; Add 5.9 mL of glacial acetic acid (National Pharmaceutical Reagent, analytical grade) to 1 L of water and stir evenly with a glass rod to prepare a 0.1 N solution B for later use; Take 192 mL of solution A and add 12 mL of solution B to prepare 200 mL of acetic acid-sodium acetate buffer (hereinafter referred to as buffer) with a pH of 3.5.

[0080] (2) Preparation of hyaluronidase solution: Take hyaluronidase (activity ≥ 560 units / mg, sigma) and prepare it into 100 u / mL with buffer solution before use.

[0081] (3) Preparation of calcium chloride solution: Dissolve 0.1387 g of anhydrous calcium chloride (National Pharmaceutical Reagent, analytical grade) in 100 mL of water to prepare a 12.5 mM solution and store in a dark place.

[0082] (4) Preparation of sodium hyaluronate solution: Dissolve 0.12 g of sodium hyaluronate (Freda) in 100 mL of pH 3.5 buffer to prepare a 1.2 mg / mL solution and store in a dark place.

[0083] (5) Preparation of sodium hydroxide solution: Dissolve 0.4 g of sodium hydroxide (National Pharmaceutical Reagent, analytical grade) in 25 mL of water to prepare a 0.4 M solution and store in a dark place.

[0084] (6) Preparation of potassium borate solution: Dissolve 30.55 g of potassium tetraborate in 250 mL of water to prepare a 0.4 M solution and store in a dark place.

[0085] (7) Preparation of p-dimethylaminobenzaldehyde solution: Take 41.5 mL of concentrated hydrochloric acid and add it to 8.5 mL of water to prepare a 10 N solution C, which is set aside. Take 4 g of p-dimethylaminobenzaldehyde and dissolve it in 350 mL of acetic acid and 50 mL of solution C (note that acetic acid should be added first and then hydrochloric acid) to prepare a 10 mg / ml solution, which is stored in the dark.

[0086] (8)Perform the operations as shown in the table below.

[0087] Table 1

[0088] T <![CDATA[T0]]> C <![CDATA[C0]]> Example or positive control (μL) 400 400 — — Buffer (μL) — 50 400 450 Hyaluronidase (μL) 50 — 50 — Number of parallels 3 1 3 1

[0089] Use 10mL test tubes to set up four groups, T, T0, C, and C0. Three parallels are required for each tested concentration of the T group of the example or positive control, and three parallels are required for the C group. 400 μL of sample solution of the same concentration is added to each of the T group and the T0 group, and 400 μL of buffer is added to each of the C and C0 groups. 50 μL of hyaluronidase solution is added to each of the T and C groups, and 50 μL of buffer is used instead of the T0 and C0 groups. The example or positive control and hyaluronidase are fully mixed and placed in a 37°C water bath for 20 minutes.

[0090] Add 100 μL of 12.5 mM calcium chloride solution to each test tube and incubate in a 37°C water bath for 20 min.

[0091] Add 250 μL of 1.2 mg / mL sodium hyaluronate solution to each test tube and incubate in a 37°C water bath for 40 min.

[0092] Add 100 μL of 0.4 N sodium hydroxide solution and 100 μL of 0.4 M potassium borate to each test tube, place in a boiling water bath for 3 min, and cool to room temperature.

[0093] Add 3 mL of 10 mg / mL p-dimethylaminobenzaldehyde to the test tube, incubate in 37°C water bath for 20 min, place in a cuvette, and measure the OD value at 585 nm.

[0094]

[0095] Wherein T is the embodiment or positive control group; T0 is the embodiment or positive control background group; C is the enzyme reaction group; C0 is the solvent background group.

[0096] 2. Experimental results:

[0097] Table 2

[0098]

[0099]

[0100] The hyaluronidase inhibition experiment showed that purpurogenol had a good hyaluronidase inhibition effect in the mass concentration range of 2.225μg / mL to 17.8μg / mL. At the same time, the hyaluronidase inhibition rate IC50 of purpurogenol was 4.9μg / mL, which was better than the hyaluronidase inhibition rate IC50 of sodium cromoglycate (51.9μg / mL).

[0101] Example 6: Evaluation of the effect of porphyrin on macrophages

[0102] This test stimulates macrophages with LPS, detects changes in the levels of pro-inflammatory factors (IL-6, TNF-α) and inflammatory mediators (PGE2) after the sample is exposed, and evaluates the soothing effect of the sample to be tested.

[0103] 1 Experimental methods

[0104] 1.1 Cytotoxicity assay

[0105] (1) Cell seeding: 9×10 3 The cells were seeded into a 96-well plate at a seeding density of 100 cells / well and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0106] (2) Experimental grouping: The experiment set up a zero adjustment group, a solvent control group, a positive control group and Example 1. In the sample group, 8 concentration gradients were set for each sample, and 3 replicate wells were set under each concentration gradient.

[0107] (3) Solution preparation: Prepare working solutions of the test substance of different concentrations according to the test concentration setting table (Table 3).

[0108] Table 3

[0109]

[0110] (4) Administration: Administration was performed when the cell plating rate in the 96-well plate reached 40% to 60%. 200 μL of cell culture medium (Gibco) 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 test substance at the corresponding concentration was added to each well of the administration group; the zero well 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 (37°C, 5% CO2) and cultured for 24 hours.

[0111] (5) Detection: After the cells were incubated for 24 h, the supernatant was discarded, and MTT working solution (0.5 mg / mL, freshly prepared, Sigma) was added. The cells were incubated at 37°C in the dark for 4 h. After the incubation, the supernatant was discarded, 150 μL DMSO was added to each well, and the absorbance was measured at 490 nm. The cell survival rate was calculated. The results were expressed as Mean ± SD.

[0112] (6) Calculation formula:

[0113] 1.2 Experimental Results

[0114] Table 4

[0115]

[0116] The results of cytotoxicity test show that when the mass concentration of purpurogenol is 0.278125 μg / mL, the cell survival rate is 89.79%. Therefore, according to the MTT results, purpurogenol has no cytotoxicity within the concentration range of 0.278125 μg / mL.

[0117] 1.3 Determination of IL-6, TNF-α and PGE2 content

[0118] (1) Cell seeding: 2.2×10 5 Cells were inoculated into 6-well plates at a density of 100 cells / well, 2 mL of cell suspension was added to each well, and the plates were incubated in an incubator (37° C., 5% CO 2 ) for 24 h.

[0119] (2) Solution preparation: divided into blank control group, negative control group, dexamethasone group (0.1 μg / mL) and Example 1 group (0.278125 μg / mL, 0.1390625 μg / mL and 0.06953125 μg / mL).

[0120] (3) Administration: Aspirate and discard the old cell culture medium in the wells, then add 1.8 mL of culture medium containing the test substance at the corresponding concentration to each well of the administration group; add 1.8 mL of solvent control culture medium to each well of the negative control group; add 1.8 mL of positive control culture medium to each well of the positive control group; and add 1.8 mL of culture medium to each well of the blank control group. Then, place the 6-well plate in an incubator (37°C, 5% CO2) and incubate for 2 h.

[0121] (4) LPS stimulation: 2 h after administration, 200 μL of LPS-containing working solution was added to each well of each group and the cells were incubated in an incubator (37° C., 5% CO 2 ) for 22 h.

[0122] (5) ELISA detection (mouse IL-6 ELISA kit, manufacturer: Abcam; mouse TNF alpha ELISA kit, manufacturer: Abcam; Prostaglandin E2 ELISA kit manufacturer: Abcam): collect cell culture supernatant and detect IL-6, TNF-α, and PGE2 according to the ELISA kit instructions.

[0123] (6) Statistical analysis of results: The results were expressed as Mean ± SD. Statistical analysis was performed using t-test, with P < 0.05 considered to be significantly different and P < 0.01 considered to be extremely significantly different.

[0124] 1.4 IL-6, TNF-α, and PGE2 content test results

[0125] Table 5

[0126]

[0127] 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 *.

[0128] Compared with the blank control group, the IL-6 concentration in the negative control group increased from 2.34 pg / mL to 353.10 pg / mL, indicating that the stimulation conditions of this test were effective.

[0129] Compared with the negative control group, the IL-6 concentration of the positive control group (dexamethasone) decreased from 353.10pg / mL to 125.74pg / mL, and the inhibition rate reached 64.39%, indicating that the positive control of this test was effective. When the mass concentration of purpurogenol was 0.1390625μg / mL, the IL-6 concentration decreased from 353.10pg / mL to 283.87pg / mL, and the inhibition rate reached 19.61%; when the mass concentration was 0.278125μg / mL, the IL-6 concentration decreased from 353.10pg / mL to 187.62pg / mL, and the inhibition rate reached 46.86%. This shows that purpurogenol can effectively inhibit the expression of IL-6.

[0130] Table 6

[0131]

[0132] Compared with the blank control group, the TNF-α concentration in the negative control group increased from 1097.77 pg / mL to 27612.49 pg / mL, indicating that the stimulation conditions of this test were effective.

[0133] Compared with the negative control group, the TNF-α concentration of the positive control group (dexamethasone) decreased from 27612.49 pg / mL to 20146.49 pg / mL, and the inhibition rate reached 27.04%, indicating that the positive control in this test was effective. When the mass concentration of purpurogenol was 0.06953125μg / mL, the concentration of TNF-α decreased from 27612.49pg / mL to 21743.04pg / mL, and the inhibition rate was 21.26%; when the mass concentration was 0.1390625μg / mL, the concentration of TNF-α decreased from 27612.49pg / mL to 20146.48pg / mL, and the inhibition rate was 27.04%; when the mass concentration was 0.278125μg / mL, the concentration of TNF-α decreased from 27612.49pg / mL to 19617.65pg / mL, and the inhibition rate was 28.95%. This shows that purpurogenol can effectively inhibit the expression of TNF-α.

[0134] Table 7

[0135]

[0136] Compared with the blank control group, the PGE2 concentration in the negative control group increased from 1020.55 pg / mL to 37646.05 pg / mL, indicating that the stimulation conditions of this test were effective.

[0137] Compared with the negative control group, the PGE2 concentration of the positive control group (dexamethasone) decreased from 37646.05 pg / mL to 8758.47 pg / mL, and the inhibition rate reached 76.73%, indicating that the positive control in this test was effective. When the mass concentration of purpurogenol was 0.06953125μg / mL, the concentration of PGE2 decreased from 37646.05pg / mL to 20339.92pg / mL, and the inhibition rate was 45.81%; when the mass concentration was 0.1390625μg / mL, the concentration of PGE2 decreased from 37646.05pg / mL to 15615.19pg / mL, and the inhibition rate was 58.52%; when the mass concentration was 0.278125μg / mL, the concentration of PGE2 decreased from 37646.05pg / mL to 8645.64pg / mL, and the inhibition rate was 77.03%. This shows that purpurogenol can effectively inhibit the expression of PGE2.

[0138] From the test results of IL-6, TNF-α and PGE2 contents, it can be seen that purpurogenol can significantly inhibit the content of inflammatory mediator PGE2 at a mass concentration of 0.06953125μg / mL~0.278125μg / mL; at a mass concentration of 0.1390625μg / mL~0.278125μg / mL, it can significantly inhibit the pro-inflammatory factors IL-6 and TNF-α, indicating that purpurogenol has good soothing effect.

[0139] Osorbitolone can be used for the preparation of skin topical preparations, medicines and health foods. 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 0.0000069%-10% (w / w). The preferred weight percentage is 0.0000069%-5% (w / w). The more preferred weight percentage is 0.000013%-1% (w / w). The most preferred weight percentage is 0.000027%-0.5% (w / w).

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

[0141] Application Example 1: Preparation of facial cream

[0142]

[0143] Application Example 2: Preparation of Emulsion

[0144]

[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]

[0154] Application Example 6: Preparation of facial mask

[0155]

[0156]

[0157] Application Example 7: Preparation of Eye Cream

[0158]

[0159] Application Example 8: Preparation of Aerosol (Cleaning Bubble)

[0160]

[0161]

[0162] Application Example 9: Preparation of Spray

[0163]

[0164] Application Example 10: Preparation of shower gel

[0165]

[0166]

[0167] Application Example 11: Preparation of facial cleanser

[0168]

[0169]

Claims

1. Anti-inflammatory and soothing applications of porphyrin.

2. The use according to claim 1, wherein: The anti-inflammatory relief is achieved by inhibiting the activity of hyaluronidase.

3. The use according to claim 1, wherein: The anti-inflammatory relief is achieved by inhibiting IL-6, TNF-α and / or PGE2.

4. The use according to claim 1, characterized in that The concentration of porphyrone used is at least 0.1 μg / mL.

5. Application of porphyrin in the preparation of skin topical preparations, medicines and health foods with anti-inflammatory and soothing effects.

6. The use according to claim 5, wherein: The anti-inflammatory relief is achieved by inhibiting the activity of hyaluronidase.

7. The use according to claim 5, wherein: The anti-inflammatory relief is achieved by inhibiting IL-6, TNF-α and / or PGE2.

8. 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.

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