CN117982360A - Anti-inflammatory and soothing application of desmethoxy pod pterosin - Google Patents

Abstract

The invention provides an anti-inflammatory and soothing application of demethoxy legumin. The anti-inflammatory relief of the present invention may be achieved by inhibiting the activity of hyaluronidase. Anti-inflammatory relief may also be achieved by inhibiting IL-6 and/or PGE 2. The invention also relates to the application of the demethoxy legumin in preparing external skin preparations, medicines and health-care foods with anti-inflammatory and soothing effects, wherein the external skin preparations are selected from the following components: face cream, milky lotion, jelly, pack, face toilet, pack, aerosol cleansing foam, spray, body wash, or facial cleanser.

Description

Anti-inflammatory and soothing application of desmethoxy pod pterosin

Technical Field

The invention relates to the field of natural pharmaceutical chemistry, in particular to anti-inflammatory and soothing effects of demethoxy pod pterosin and application of the demethoxy pod pterosin in preparing skin external preparations, medicines and health-care foods with the anti-inflammatory and soothing effects.

Background

Hyaluronic Acid (HA), commonly known as hyaluronic acid, is a high molecular weight linear macromolecular acidic mucopolysaccharide, and is formed by repeatedly and alternately connecting double pond units of D-glucuronic acid and N-acetylamino-D-glucose. Natural HA is widely present in various tissues in higher animals, but in varying amounts. Which is mainly distributed in the cell matrix and lubricating fluid. HA is used as a multifunctional matrix in a organism, and HAs important physiological functions of regulating and controlling cell proliferation, differentiation, migration, lubricating joints, protecting cartilage, promoting wound healing, resisting oxidation, resisting aging and the like. HA HAs extremely strong water-retaining effect, and the water-retaining effect is higher than that of other water-retaining substances found in the prior nature, is an ideal natural water-retaining factor, and HAs been widely applied to the fields of clinical medicine, cosmetic production and the like. Hyaluronidase is a glycosidase that primarily degrades hyaluronic acid. Under normal conditions, hyaluronic acid and hyaluronidase in the body are in an equilibrium state, and once the activity of the hyaluronidase is too high, the hyaluronic acid is excessively degraded, and the equilibrium is broken, so that various diseases can be caused to occur and develop.

Interleukin-6 (interleukin-6, IL-6) is a multifunctional, multipotent and multipotent cytokine secreted by T cells, B cells, monocytes, macrophages, fibroblasts, vascular endothelial cells and some tumor cells, and is one of the most functional cytokines to date. Dysfunction and abnormal expression of IL-6 are typical features of certain diseases. In tumor cells, IL-6 is involved in the proliferation, differentiation, invasion, metastasis, vascular proliferation and other processes of tumor cells through a plurality of signal pathways. The rise of serum IL-6 level and the abnormal expression of the receptor thereof are closely related to the occurrence and development of various tumors, such as lung cancer, gastric cancer, bladder cancer, liver cancer and the like. IL-6 is also related to the nervous system and has been shown by foreign studies. IL-6 is also associated with neuropathic pain. IL-6 disrupts the balance between Treg cells and Th17 cells and promotes the development of psoriasis. IL-6 also promotes clotting without affecting fibrinolysis, ultimately leading to thrombosis. Therefore, inhibition of excessive secretion of IL-6 can inhibit the occurrence and development of various diseases.

Prostaglandin E2 (prostaglandin E, PGE 2) is a very important lipid metabolite in the body that is released when subjected to various physiological or pathological stimuli, which in turn leads to the development of the associated disease. PGE2 is involved in many tumor-promoting steps, including accelerating tumor cell proliferation, anti-tumor cell apoptosis, enhancing tumor cell invasion and metastasis, promoting angiogenesis, etc. Therefore, the production of PGE2 is inhibited, and the occurrence and development of diseases such as tumors are inhibited.

Desmethoxy pod fern (5, 7-dihydroxy-6, 8-dimethoxy flavanone), CAS no: 56297-79-1, english name: demethoxymatteucinol, molecular formula: c ₁₇H₁₆O₄, molecular weight: 284.31, the structural formula is as follows:

Desmethoxy fern is a flavonoid component and widely exists in plants such as rhizoma Cyrtomii Falcati, herba Botrytis sinensis, herba Drynariae Latifoliae, and guava. Among the cosmetic related efficacy reports, only a small amount of desmethoxy fagin was studied for antioxidant effect, and no cosmetic report was found on anti-inflammatory and soothing effect of desmethoxy fagin.

The invention surprisingly discovers that the demethoxy legumin has better hyaluronidase activity inhibition, IL-6 and PGE2 generation reduction and anti-inflammatory and soothing effects.

Disclosure of Invention

In one aspect, the present invention relates to the anti-inflammatory soothing use of desmethoxy pod pterosin.

In preferred embodiments, the anti-inflammatory relief is achieved by inhibiting hyaluronidase activity and/or by inhibiting IL-6 and/or PGE 2.

In a preferred embodiment, desmethoxy-pod fern is used at a concentration of at least 0.1 μg/mL, preferably 0.1-28.4 μg/mL, more preferably 0.445-14.2 μg/mL.

On the other hand, the invention also relates to the application of the demethoxy legumin in preparing external skin preparations, medicines and health-care foods with anti-inflammatory and soothing effects.

In a preferred embodiment, the content of desmethoxy-pod pterosin in skin external preparations, medicines and health foods is 0.00001-20 wt%, preferably 0.0001-10 wt%.

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.

Detailed Description

The invention discovers that the demethoxy pod pteridon has better effects of inhibiting the activity of hyaluronidase, reducing the generation of IL-6 and PGE2, resisting inflammation and relieving, and can be used as an effective component to be added into skin external preparations, medicines and health-care foods for use so as to help to improve skin redness, tingling, allergic problems 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 desmethoxy-pod pterosin described herein may 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 desmethoxy pod fern. The product may additionally have a package such as a plastic or cardboard box for storing the container. In one embodiment, the product comprises desmethoxy-pod fern and has instructions for directing the user to apply the desmethoxy-pod fern 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.

Desmethoxy pod pterosin

The present invention is based on the following unexpected findings: desmethoxy pod pterosin has antiinflammatory and relieving effects. Therefore, the demethoxy pod pteridon can be used as an effective component to be added into skin external preparations, medicines and health-care foods to assist in improving skin redness, tingling, allergy problems and the like.

In anti-inflammatory relief applications, desmethoxy pod fern is used at a concentration of at least 0.001mg/mL, preferably at least 0.01mg/mL.

In some embodiments, desmethoxy pod pterosin is used at a concentration of 0.0036mg/mL to 0.0284mg/mL. In some embodiments, desmethoxy pod pterosin is used at a concentration of 0.0071mg/mL to 0.0284mg/mL, more preferably 0.0142mg/mL to 0.0284mg/mL.

In some embodiments, desmethoxy pod fern is used at a concentration of at least 0.1 μg/mL, preferably at least 1 μg/mL. In some embodiments, desmethoxy pod pterosin is used at a concentration of 0.445 μg/mL to 14.2 μg/mL.

External preparation for skin

In some embodiments, desmethoxy pod pterosin may be used in the preparation of external skin preparations. The skin external preparation is preferably a cosmetic composition including, but not limited to, products in the form of face cream, milky lotion, jelly, lotion, essence, pack, eye cream, aerosol (cleansing foam), spray, body wash, facial cleanser, and the like.

The content of desmethoxy-pod fern in the skin external preparation is 0.0001-20wt%, preferably 0.001-10wt%, more preferably 0.01-5wt%.

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 external preparation for skin comprising desmethoxy-pod fern 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 external preparation for skin containing desmethoxy pod pterosin can be formulated into solution. The solution typically comprises an aqueous or organic solvent (e.g., about 50% to about 99.99% or about 90% to about 99% of a cosmetically acceptable aqueous or organic solvent). Examples of suitable organic solvents include propylene glycol, polyethylene glycol, polypropylene glycol, glycerol, 1,2, 4-butanetriol, sorbitol esters, 1,2, 6-hexanetriol, ethanol and mixtures thereof.

The skin external preparation may be formulated as a solution containing an emollient. Such skin external preparations preferably comprise from about 2% to about 50% of one or more emollients. As used herein, "emollient" refers to a substance used to prevent or reduce dryness, for example, by preventing the loss of skin moisture through the skin. Examples of emollients include, but are not limited to, vegetable oils, mineral oils, aliphatic esters, and the like.

Lotions can be prepared from such solutions. Lotions typically contain from about 1% to about 20% (e.g., from about 5% to about 10%) of one or more emollients and from about 50% to about 90% (e.g., from about 60% to about 80%) of moisture.

Another type of product that can be formulated from solutions is a cream. A cream typically contains from about 5% to about 50% (e.g., from about 10% to about 20%) of one or more emollients and from about 45% to about 85% (e.g., from about 50% to about 75%) of moisture.

Although it is preferred that the skin external preparation comprising desmethoxy-pod fern comprises water, the skin external preparation may alternatively be anhydrous or ointments that do not comprise water but instead are organic and/or silicone solvents, oils, fats and waxes. Ointments may contain simple bases of animal or vegetable oils or semi-solid hydrocarbons. Ointments may contain from about 2% to about 10% of one or more emollients and from about 0.1% to about 2% of one or more thickeners.

The skin external preparation can be formulated as an emulsion. If the topical carrier is an emulsion, from about 1% to about 10% (e.g., from about 2% to about 5%) of the topical carrier contains one or more emulsifying agents. The emulsifier may be nonionic, anionic or cationic. Examples of suitable emulsifiers include those commonly identified as suitable emulsifiers in the personal care and cosmetic formulations arts.

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

Oil-in-water and water-in-oil single emulsion skin care formulations, such as lotions and creams, are well known in the cosmetic arts and can be used in the present invention. Multiple emulsion skin external preparations (e.g., water-in-oil-in-water and oil-in-water) are also useful in the present invention. Typically, such single-phase or multiple-phase emulsions contain moisture, emollients, and emulsifiers as their essential ingredients.

The skin external preparation comprising desmethoxy-pod fern 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.

External preparations for the skin containing desmethoxy-pod fern may also be formulated as solid preparations (e.g., wax-based sticks, bar soaps, powders, or wipes containing powders).

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, and the like, as long as they are physically and chemically compatible with the other components of the skin external preparation and do not affect the effectiveness of the desmethoxyl legumins 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 treat 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 demethoxypod pterosin (purity is more than or equal to 95%) used in the embodiment of the invention is provided by Chinese medical institute of traditional Chinese medicine.

Example 1: preparation of desmethoxy pod fern

2.84Mg of desmethoxy fagin was weighed and fixed in a 10mL volumetric flask with dimethylsulfoxide (DMSO, sigma) to give 0.284mg/mL of desmethoxy fagin solution for use.

Example 2: preparation of desmethoxy pod fern

14.2Mg of desmethoxy fagin is weighed, and the volume is fixed to a 10mL volumetric flask by using DMSO to obtain 1.42mg/mL of desmethoxy fagin solution for standby.

Example 3: preparation of desmethoxy pod fern

0.0313ML of the desmethoxy pod fern solution of example 2 was taken, and the volume was set to a 100mL volumetric flask using DMEM medium (Gibco) to obtain 0.445. Mu.g/mL of the desmethoxy pod fern solution for use.

Example 4: preparation of desmethoxy pod fern

0.125ML of the desmethoxy-pod pterosin solution of example 2 is taken, the volume is fixed to a 100mL volumetric flask by using DMEM medium, and 1.775 mug/mL of the desmethoxy-pod pterosin solution is obtained for later use.

Example 5: preparation of desmethoxy pod fern

1ML of the desmethoxy-pod pterosin solution of example 2 was taken, and the volume was fixed to a 100mL volumetric flask with DMEM medium to obtain 14.2. Mu.g/mL of the desmethoxy-pod pterosin solution for use.

Test example 1: in vitro hyaluronidase Activity inhibition assay

High molecular weight hyaluronic acid plays an important role in regulating scar-free repair of wound healing, and can significantly reduce inflammatory response. However, degradation products of hyaluronic acid can increase inflammatory response in wound healing process, and the degradation reaction of hyaluronic acid depends on hyaluronidase activity, and inhibition of hyaluronidase activity can be used as index of anti-inflammatory response of substances. Hyaluronidase can hydrolyze substrate hyaluronic acid under acidic environment, and convert into N-acetylglucosamine. N-acetylglucosamine and dimethylaminobenzaldehyde produce a color reaction, and the activity of hyaluronidase is determined according to the color intensity.

1. Test sample

Samples 1-4 were obtained from the desmethoxy pod pterosin solution of example 1 by volume fraction dilution with acetic acid-sodium acetate buffer at pH 3.5. Table 1 shows the information of the samples to be tested prepared in example 1.

TABLE 1

	Sample 1	Sample 2	Sample 3	Sample 4
					Volume fraction (V/V)	100％	50％	25％	12.5％
Concentration (mg/mL)	0.284	0.142	0.071	0.036

2. Experimental method

(1) Buffer solution with pH of 3.5: 1.3609g of sodium acetate trihydrate (national medicine reagent, analytical grade) is taken and dissolved in 100mL of water, and is uniformly stirred by a glass rod to prepare 0.1M solution A for later use; adding 5.9mL of glacial acetic acid (Chinese medicinal reagent, analytically pure) into 1L of water, and uniformly stirring with a glass rod to prepare 0.1N solution B for later use; 192mL of the solution A was added to 12mL of the solution B to prepare 200mL of an acetic acid-sodium acetate buffer (hereinafter referred to as buffer) having a pH of 3.5.

(2) Preparing hyaluronidase solution: hyaluronidase (activity is more than or equal to 560unit/mg, sigma) is prepared into 100u/mL by buffer solution for clinical preparation.

(3) Preparing a calcium chloride solution: 0.1387g of anhydrous calcium chloride (national reagent, analytical grade) was dissolved in 100mL of water to prepare a 12.5mM solution, which was stored in the dark.

(4) Preparing sodium hyaluronate solution: 0.12g of sodium hyaluronate (Furita) was dissolved in 100mL of buffer pH 3.5 to prepare a 1.2mg/mL solution, which was stored in the dark.

(5) Preparing sodium hydroxide solution: 0.4g of sodium hydroxide (national drug reagent, analytical grade) was dissolved in 25mL of water to prepare a 0.4M solution, which was stored in the dark.

(6) Preparing a potassium borate solution: 30.55g of potassium tetraborate is dissolved in 250mL of water to prepare a 0.4M solution, and the solution is stored in a dark place.

(7) Preparing a p-dimethylaminobenzaldehyde solution: adding 41.5mL of concentrated hydrochloric acid into 8.5mL of water to prepare 10N solution C for later use; 4g of p-dimethylaminobenzaldehyde was dissolved in 350mL of acetic acid and 50mL of solution C (note that acetic acid was added first followed by hydrochloric acid) to prepare a 10mg/mL solution, which was stored in the dark.

(8) Taking a 10mL test tube as a reaction tube, adding 400 mu L of sample solution with corresponding concentration into a sample tube (T) and a sample control tube (T0), and replacing a blank tube (C) and a blank control tube (C0) by a Buffer; 50 mu L of hyaluronidase solution (activity is more than or equal to 560unit/mg, sigma) is added into the sample tube (T) and the blank tube (C) and uniformly mixed, 50 mu L of Buffer is added into the sample control tube (T0) and the blank control tube (C0) and uniformly mixed, and water bath is carried out for 20min at 37 ℃.

The reaction tubes were each charged with 100. Mu.L of 12.5mM calcium chloride in a water bath at 37℃for 20min.

The reaction tubes were each charged with 250. Mu.L of sodium hyaluronate in a water bath at 37℃for 40min.

The reaction tubes were each charged with 100. Mu.L of 0.4N sodium hydroxide and 100. Mu.L of 0.4M potassium borate, and cooled to room temperature by a boiling water bath for 3 min.

The reaction tubes were sequentially charged with 3mL of p-dimethylaminobenzaldehyde in a water bath at 37℃for 20min (the reaction time of each reaction tube in this step was kept exactly the same). The solution in each reaction tube was transferred to a cuvette and absorbance (OD) was measured at 585nm in a spectrophotometer.

3. Experimental results:

Samples 1-4 were converted to final test concentrations according to the reaction system, i.e., 0.0284mg/mL, 0.0142mg/mL, 0.0071mg/mL, 0.0036mg/mL.

Results are expressed as mean±sd. Table 2 shows the results of inhibition of the hyaluronidase activity of desmethoxy-pod fern in vitro.

TABLE 2

Final detection concentration (mg/mL)	Hyaluronidase inhibition rate (%)
		0.0284	92.39
0.0142	82.61
		0.0071	30.43
0.0036	5.43

As is clear from Table 2, desmethoxy-pod fern was able to significantly inhibit hyaluronidase activity in the concentration range of 0.0036mg/mL to 0.0284mg/mL, with increasing concentration, the inhibition rate increased, and there was a concentration dependence.

Test example 2: IL-6 test results

The test is to test the relief efficacy of the sample to be tested by stimulating macrophages with LPS and detecting the change of the content of pro-inflammatory factors (IL-6) after the sample acts.

1. Test packets

The experiments were divided into a Blank (BC), a Negative (NC) and a sample (examples 3-5).

2. Experimental method

(1) Cell inoculation: macrophages (210622, boxi organisms) were inoculated into 6-well plates at an inoculation density of 2.2X10 ⁵/well and incubation (5% CO ₂, 37 ℃) continued for 24h in an incubator.

(2) Administration: old cell culture fluid in the wells was aspirated. 1.8mL of culture solution containing the test substance with corresponding concentration is added into each hole of the sample group; adding 1.8mL of solvent control culture solution into each well of NC group; 1.8mL of normal culture solution was added to each well of BC group. After the completion of the administration, the 6-well plate was placed in a cell incubator (5% CO ₂, 37 ℃) and incubated for 2 hours.

(3) LPS stimulation: 200. Mu.L of working solution containing LPS (E. Coli. Sigma) was added to each well of NC group and sample group, and the mixture was placed in a cell incubator (5% CO ₂, 37 ℃) for further culture for 22 hours.

(4) ELISA detection: cell culture supernatants were collected and assayed by ELISA according to the IL-6ELISA kit (Abcam) instructions.

(5) Analysis of results: results are expressed as mean±sd. The IL-6 test results are shown in Table 3.

TABLE 3 Table 3

Experimental grouping	IL-6 average concentration (pg/mL)
		BC	2.34±0.15
NC	353.10±19.75
		Example 3	227.14±13.14
Example 4	335.85±19.78
		Example 5	357.41±12.92

The increase in IL-6 concentration from 2.34pg/mL to 353.10pg/mL in the Negative Control (NC) compared to BC indicates that the stimulation conditions of this test are effective.

The IL-6 content of example 3 (0.445. Mu.g/mL desmethoxy pod pterosin solution) was reduced from 353.10pg/mL to 227.14pg/mL compared to NC group; the decrease in IL-6 content of example 4 (1.775. Mu.g/mL of desmethoxy-pod fern solution) from 353.10pg/mL to 335.85pg/mL indicates that desmethoxy-pod fern is very effective in inhibiting IL-6 secretion.

Test example 3: PGE2 test results

According to the test, the LPS is adopted to stimulate macrophages, the change condition of the content of inflammatory mediators (PGE 2) after the sample acts is detected, and the relieving effect of the sample to be tested is evaluated.

1. Test packets

The experiments were divided into a Blank (BC), a Negative (NC) and a sample (examples 3-5).

2. Experimental method

(1) Cell inoculation: macrophages (210622, boxi organisms) were inoculated into 6-well plates at an inoculation density of 2.2X10 ⁵/well and incubation (5% CO ₂, 37 ℃) continued for 24h in an incubator.

(2) Administration:

old cell culture fluid in the wells was aspirated. 1.8mL of culture solution containing the test substance with corresponding concentration is added into each hole of the sample group; adding 1.8mL of solvent control culture solution into each well of NC group; 1.8mL of normal culture solution was added to each well of BC group. After the completion of the administration, the 6-well plate was placed in a cell incubator (5% CO ₂, 37 ℃) and incubated for 2 hours.

(3) LPS stimulation: 200. Mu.L of working solution containing LPS (E. Coli. Sigma) was added to each well of NC group and sample group, and the mixture was placed in a cell incubator (5% CO ₂, 37 ℃) for further culture for 22 hours.

(4) ELISA detection: cell culture supernatants were collected and assayed by ELISA according to the PGE2 ELISA kit (Abcam) instructions.

(5) Analysis of results: results are expressed as mean±sd. Table 4 shows PGE2 test results.

TABLE 4 Table 4

Experimental grouping	PGE2 average concentration (pg/mL)
		BC	1020.55±42.09
NC	37646.05±2452.79
		Example 3	30078.68±1144.24
Example 4	26106.79±1322.81
		Example 5	3502.33±179.72

The increase in PGE2 concentration from 1020.55pg/mL to 37646.05pg/mL in the Negative Control (NC) compared to the Blank (BC) demonstrates that the stimulation conditions of this test are effective.

In comparison to the NC group, the PGE2 content of example 3 (0.445. Mu.g/mL of desmethoxy-pod fern solution) was decreased from 37646.05pg/mL to 30078.68pg/mL, the PGE2 content of example 4 (1.775. Mu.g/mL of desmethoxy-pod fern solution) was decreased from 37646.05pg/mL to 26106.79pg/mL, and the PGE2 content of example 5 (14.2. Mu.g/mL of desmethoxy-pod fern solution) was decreased from 37646.05pg/mL to 3502.33pg/mL. The PGE2 content is obviously reduced along with the increase of the concentration of the demethoxy legumin, which indicates that the inhibition effect is enhanced along with the increase of the concentration of the demethoxy legumin and a certain concentration dependence is presented. Indicating that the desmethoxy pod pterosin can well inhibit PGE2 secretion.

The desmethoxy pod pterosin can be used for preparing skin external preparation, medicine and health food. The skin external preparation is preferably a cosmetic composition, including but not limited to preparation of products in the forms of face cream, emulsion, gel, lotion, essence, facial mask, eye cream, aerosol (cleansing foam), spray, shower gel, facial cleanser and the like. The weight percentage of the desmethoxy pod fern in the skin external agent is 0.00001% -20% (w/w). Preferably 0.0001% to 10% (w/w). More preferably 0.001% to 5% (w/w). Most preferably 0.01% -5% (w/w) by weight.

The following are specific examples of application of desmethoxy-pod pterosin in skin external preparations, and formulations and preparation methods of the formulations. Specific applications are as follows: application example 1: preparation of face cream

Application example 2: preparation of the emulsion

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Application example 3: preparation of jelly

Application example 4: preparation of toning lotion

Application example 5: preparation of essence

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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

Application example 11: preparation of facial cleanser

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Claims (10)

1. Anti-inflammatory and soothing application of desmethoxyl pod pterosin is provided.

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 inhibition of IL-6 and/or PGE 2.

4. Use according to claim 1, wherein the desmethoxyl pod pterosin is used in a concentration of at least 0.1 μg/mL, preferably 0.1-28.4 μg/mL, more preferably 0.445-14.2 μg/mL.

5. Application of desmethoxy pod fern in preparing skin external preparation, medicine and health food with antiinflammatory and relieving effects is provided.

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 inhibition of IL-6 and/or PGE 2.

8. The use according to claim 5, wherein the desmethoxy-pod fern is present in the skin external preparation, the medicament or the health food in an amount of 0.00001-20 wt%.

9. The use according to claim 8, wherein the desmethoxyl pod pterosin is contained in an amount of 0.0001-10% by weight in external preparations for skin, medicines and health foods.

10. The use according to claim 5, 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.