Anti-saccharification application of arteannuic acid

Abstract

The invention provides anti-saccharification application of arteannuic acid. The anti-saccharification of the present invention is an end product that inhibits non-enzymatic glycosylation reactions. The anti-saccharification of the invention may also be the inhibition of glycosylation end products. The invention also relates to an application of arteannuic acid in preparation of an external skin preparation, a medicine and a health-care food with an anti-saccharification effect. The external skin preparation is selected from face cream, emulsion, gel, toning lotion, essence, a mask, eye cream, aerosol cleaning foam, spray, shower gel or facial cleanser.

Description

Technical Field

[0001] The present invention relates to the field of natural drug chemistry, and in particular to the anti-glycation application of artemisinic acid and the application of artemisinic acid in the preparation of skin external preparations, medicines and health foods with anti-glycation effects. Background Art

[0002] As we age, our metabolism slows down, life and work pressures increase, and with irregular lifestyles and eating habits, the sugar we consume will not be metabolized immediately and will accumulate in the body. In the absence of enzymes, free sugars react with collagen in the dermis to form some reversible primary "glycosylation products", which then form highly fluorescent, cross-linked and irreversible brown non-enzymatic glycation end products (AGEs). The whole process is called non-enzymatic glycosylation (NEG), and AGEs can further directly cross-link biological macromolecules in the body, causing functional damage.

[0003] When elastin and collagen in the skin participate in non-enzymatic glycation reactions to generate AGEs, a cross-linked product of collagen and elastin is formed. As collagen cross-linking gradually increases, the solubility of collagen decreases, insoluble collagen accumulates, and the permeability of skin tissue decreases, resulting in barriers to the exchange of nutrients and metabolites between cells, which ultimately leads to decreased skin elasticity, sagging, and wrinkles. In addition, glycated collagen turns yellow and becomes brittle, so as we age, AGEs accumulate in large quantities and the skin gradually turns yellow. In addition, when the receptors of AGEs (AGER) are activated, they induce the body to produce a large number of free radicals, which adds a large burden to the antioxidant system in the body, further damages the skin tissue, stimulates the production of melanin, and puts the skin into a vicious cycle. Therefore, in recent years, the anti-glycation theory has become a hot mechanism and target for skin beauty. The anti-glycation effect is not only closely related to skin aging, but also closely related to the dark yellow color of the skin.

[0004] At present, the representative anti-glycation ingredients include aminoguanidine, carnosine, lipoic acid, niacinamide, quercetin and curcumin, etc. The mechanism is mainly to block the development of AGEs and inhibit the generation of AGEs. Among them, aminoguanidine is a drug known to effectively prevent the glycation process, and carnosine is a recognized effective anti-glycation ingredient in the skin field.

[0005] According to searches in recent years, Chinese patent application 201710075645.5 discloses an anti-glycation mask containing Poria cocos extract. Chinese patent application 201810568900.4 discloses an anti-glycation black tea extract and a preparation method thereof. Chinese patent application 201911365050.9 discloses a method for evaluating the anti-glycation effect of plant extracts. Chinese patent application 202011130862.8 discloses a brightening and anti-glycation composition containing carnosine and ergothioneine and its application. At the same time, there are many other patents for anti-glycation compositions, involving bamboo sap extract, Huoshan dendrobium, golden thread lotus extract, ginkgo leaf extract, etc. However, none of these documents involve anti-glycation research on artemisinin compounds. The research on compound monomers extracted and separated from natural Chinese herbal medicines in the field of anti-glycation is almost blank, and it may become a hot spot for exploration in the future.

[0006] Artemisinic acid, English name Artemisinic acid, CAS number 80286-58-4, molecular weight: 234.33, molecular formula: C15H22O2, artemisinic acid structure formula is as follows. Artemisinic acid boiling point 373.6 ± 11.0 ℃ (760mmHg), melting point 129-131 ℃, soluble in methanol, ethanol and DMSO and other organic solvents.

[0007]

[0008] Artemisinic acid is derived from the Asteraceae plant Artemisia annua. It is a sesquiterpene isolated from Artemisia annua and has multiple pharmacological activities, such as antimalarial activity, antitumor activity, antipyretic effect, antibacterial activity, and anti-lipogenesis. In vitro studies have shown that artemisinic acid can significantly inhibit cell proliferation in human liver cancer cell SMMC-7721 cells in a time- and dose-dependent manner. After 48 hours of high-concentration artemisinic acid, SMMC-7721 cells showed typical apoptosis characteristics, with an apoptosis rate of 67.54%. This shows that artemisinic acid can inhibit the proliferation of SMMC-7721 cancer cells, which may be related to the induction of apoptosis, thus revealing the anti-tumor potential of artemisinic acid. In addition, studies have confirmed that artemisinic acid can be used as an adjuvant treatment for obesity associated with metabolic syndrome. Its mechanism of action is that artemisinic acid inhibits the adipose differentiation of human adipose tissue-derived mesenchymal stem cells (hAMSCs) by inhibiting the expression of C / EBPδ mediated by JNK.

[0009] In the literature search in the past 10 years, with artemisinic acid as the keyword, most of the existing studies have focused on the extraction and purification, synthetic preparation and content identification of artemisinic acid. There are fewer studies focusing on the biological activity of artemisinic acid. There are also few reports on its application in the fields of skin and health food, and no research on its anti-glycation aspect.

[0010] The present invention unexpectedly discovered that artemisinic acid has an anti-glycation effect and can effectively reduce the content of AGEs, the end product of non-enzymatic glycation reaction. Summary of the invention

[0011] The invention provides the anti-glycation application of artemisinic acid.

[0012] In a preferred embodiment, artemisinic acid is used at a concentration of at least 0.1 mg / L, more preferably at least 1 mg / L.

[0013] In a preferred embodiment, anti-glycation is to inhibit the end products of non-enzymatic glycosylation reactions. In a preferred embodiment, anti-glycation is to inhibit the end products of glycation.

[0014] The present invention also relates to the use of artemisinic acid in the preparation of medicines, health foods or skin external preparations with anti-glycation effects.

[0015] In a preferred embodiment, the content of artemisinic acid in the medicine, health food, or external skin preparation is 0.00001-20% by weight, more preferably 0.0001-5% 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 results of the anti-glycation test of artemisinic acid are shown.

[0019] Figure 2 The anti-glycation efficacy fluorescence staining diagram of the blank group is shown. The immunofluorescence diagram is a fluorescence image taken by a fluorescence microscope, and the image is quantitatively analyzed using Image J. Blue is DAPI staining of the cell nucleus, red is CML fluorescence staining, and the magnification is 200 times.

[0020] Figure 3 The anti-glycation efficacy fluorescence staining image of the control group is shown. The immunofluorescence image is a fluorescence image taken by a fluorescence microscope, and the image is quantitatively analyzed using Image J. Blue is DAPI staining of the cell nucleus, red is CML fluorescence staining, and the magnification is 200 times.

[0021] Figure 4 The anti-glycation efficacy fluorescence staining diagram of the 0.00117 mg / mL artemisinic acid test group is shown. The immunofluorescence image is a fluorescence image taken by a fluorescence microscope, and the image is quantitatively analyzed using Image J. The blue is DAPI staining of the cell nucleus, and the red is CML fluorescence staining, and the magnification is 200 times.

[0022] Figure 5 The anti-glycation efficacy fluorescence staining diagram of the 0.0059 mg / mL artemisinic acid test group is shown. The immunofluorescence image is a fluorescence image taken by a fluorescence microscope, and the image is quantitatively analyzed using Image J. The blue is DAPI staining of the cell nucleus, and the red is CML fluorescence staining, and the magnification is 200 times. DETAILED DESCRIPTION

[0023] The present invention relates to the anti-glycation effect of artemisinic acid, and for the first time finds that artemisinic acid can be used as a functional additive to prepare a skin external preparation to achieve skin care effects such as whitening, brightening, yellowing, and anti-aging. In some embodiments, artemisinic acid can be used as a functional additive to prepare medicines or health foods.

[0024] 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.

[0025] 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.

[0026] The artemisinic acid described herein may optionally be in finished packaging form. In one embodiment, the packaging is a container such as a plastic, metal or glass tube or jar containing artemisinic acid. The product may additionally have packaging such as a plastic or cardboard box for storing the container. In one embodiment, the product comprises artemisinic acid and has instructions for guiding the user to apply artemisinic acid to the skin to treat the skin. Such instructions may be printed on the container, a label insert, or any other packaging.

[0027] 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.

[0028] 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.

[0029] Artemisinic acid

[0030] The present invention relates to the anti-glycation effect of the compound artemisinic acid. In the study, it was found that artemisinic acid can effectively reduce the generation of AGEs, the end product of non-enzymatic glycosylation reaction, and has excellent anti-glycation effect. Therefore, artemisinic acid can be used as an effective additive for preparing medicines, health foods or skin topical preparations to achieve skin care effects such as whitening and brightening, yellowing removal, and anti-aging.

[0031] In anti-glycation applications, artemisinic acid is used at a concentration of at least 0.1 mg / L, more preferably at least 1 mg / L.

[0032] In some embodiments, artemisinic acid is used at a concentration of at least 0.001 mg / mL, more preferably at least 0.01 mg / mL.

[0033] In some embodiments, artemisinic acid is used at a concentration of 0.001-1 mg / mL. In some embodiments, artemisinic acid is used at a concentration of 0.001-0.5 mg / mL, preferably 0.001-0.005 mg / mL. In a specific embodiment, artemisinic acid is used at a concentration of 1.17 mg / L.

[0034] In some embodiments, artemisinic acid is used at a concentration of 0.005-1 mg / mL. In some embodiments, artemisinic acid is used at a concentration of 0.005-0.5 mg / mL, preferably 0.01-0.05 mg / mL. In a specific embodiment, artemisinic acid is used at a concentration of 0.0117-0.0234 mg / mL.

[0035] Medicines, health foods or skin preparations containing artemisinic acid

[0036] The artemisinic acid of the present invention can be used as an effective additive in medicines, health foods or external skin preparations.

[0037] In some embodiments, the content of artemisinic acid in the medicine, health food or skin external preparation is 0.00001-20% by weight, more preferably 0.0001-5% by weight.

[0038] In some embodiments, the drug is selected from the group consisting of tablets, capsules, emulsions, suspensions, powders, granules, solutions, and various pharmaceutical dosage forms known in the art. Different dosages are added according to different types of dosage forms.

[0039] Health food is also called functional food. It has the function of regulating human body functions, but is not for the purpose of treating diseases, and is suitable for consumption by specific groups of people. In some embodiments, the health food can be in powder form.

[0040] In some embodiments, the skin topical agent is selected from the group consisting of: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol (cleansing foam), spray, shower gel and facial cleanser. Different amounts are added according to different types of preparations.

[0041] Skin external preparations are a general concept for all ingredients that are usually applied to the outside of the skin, such as cosmetic compositions. The cosmetic compositions may include basic cosmetics, facial makeup cosmetics, body cosmetics, hair care cosmetics, etc., and there is no particular restriction on the dosage form, which can be reasonably selected according to different purposes. The cosmetic compositions may also contain different media or matrix excipients permitted by cosmetics according to different dosage forms and purposes.

[0042] The skin topical preparation comprising artemisinic acid can be topically applied to human skin and / or hair. The skin topical preparation may also include a cosmetically acceptable topical carrier, which may be about 50% by weight to about 99.99% by weight of the skin topical preparation (e.g., 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 comprise one or more selected from wetting agents, emollients, greases, moisturizers and similar substances. In one embodiment, the cosmetically acceptable topical carrier comprises a substrate such as a nonwoven fabric or a membrane material.

[0043] 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.

[0044] The skin topical preparation containing artemisinic acid 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] Although it is preferred that the skin topical preparation containing artemisinic acid contains water, the skin topical preparation may alternatively be anhydrous or an ointment containing no water but organic and / or silicone solvents, oils, 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] Skin topical preparations containing artemisinic acid can also be formulated into gels (e.g., aqueous gels, alcoholic gels, alcohol / water gels, or oily gels using suitable gelling agents). 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., hydroxymethylcellulose and hydroxypropylcellulose). 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 gels typically contain between about 0.1% and 5% by weight of such gelling agents.

[0053] The external preparation for skin application comprising artemisinic acid can also be formulated into a solid preparation (eg, a wax-based stick, a bar soap, a powder, or a wipe containing the powder).

[0054] 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.

[0055] The skin external 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 external preparation and do not affect the effect of the artemisinic acid of the present invention.

[0056] 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 %.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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 %.

[0062] 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.

[0063] Additional cosmetic active agents

[0064] 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.

[0065] 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.

[0066] 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.

[0067] Examples of hydroxy acids include, but are not limited to, glycolic acid, lactic acid, malic acid, salicylic acid, citric acid, and tartaric acid.

[0068] 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.

[0069] How to use

[0070] The skin preparation of the present invention can be topically applied to the skin of a mammal of one or more skins that need to be treated. In one embodiment, the skin preparation can be applied to the skin that needs to be treated for whitening, brightening, yellowing, and anti-aging. The skin preparation can be applied to the skin that needs such treatment according to a suitable treatment regimen, such as monthly, weekly, every other day, daily, twice a day, etc.

[0071] 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.

[0072] Example

[0073] 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.

[0074] The artemisinic acid used in the examples of the present invention was provided by the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences.

[0075] Example 1: Preparation of artemisinic acid

[0076] Weigh 0.234 g of artemisinic acid and dilute to 1 L volumetric flask with dimethyl sulfoxide (Sigma, DMSO) to obtain a 0.234 mg / mL artemisinic acid solution (i.e., 1 mM) for later use.

[0077] Example 2: Preparation of artemisinic acid

[0078] Measure 2.0 mL of the solution of Example 1, dilute it to 10 mL with DMSO to obtain a 0.0468 mg / mL artemisinic acid solution for later use.

[0079] Example 3: Preparation of artemisinic acid

[0080] 5.0 mL of the solution of Example 2 was measured and diluted to 10 mL with DMSO to obtain a 0.0234 mg / mL artemisinic acid solution for later use.

[0081] Example 4: Preparation of artemisinic acid

[0082] 5.0 mL of the solution of Example 3 was measured and diluted to 10 mL with DMSO to obtain a 0.0117 mg / mL artemisinic acid solution for later use.

[0083] Example 5: Preparation of artemisinic acid

[0084] 5.0 mL of the solution of Example 4 was measured and diluted to 10 mL with DMSO to obtain a 0.0059 mg / mL artemisinic acid solution for later use.

[0085] Example 6: Preparation of artemisinic acid

[0086] 5.0 mL of the solution of Example 5 was measured and diluted to 10 mL with DMSO to obtain a 0.0029 mg / mL artemisinic acid solution for later use.

[0087] Example 7: Preparation of artemisinic acid

[0088] 1.0 mL of the solution of Example 1 was measured and diluted to 10 mL with DMSO to obtain a 0.0234 mg / mL artemisinic acid solution. 500 μL of the above solution was measured and diluted to 10 mL with DMSO to obtain a 0.00117 mg / mL artemisinic acid solution for later use.

[0089] Example 8: Preparation of artemisinic acid

[0090] Measure 250 μL of the solution in Example 1 and dilute it to 10 mL with DMSO to obtain a 0.0059 mg / mL artemisinic acid solution for later use.

[0091] Test Example 1: Biochemical test of artemisinic acid inhibiting advanced glycation end products

[0092] 1. Test principle:

[0093] Non-enzymatic glycation (NEG) reaction, commonly known as glycation reaction, refers to the non-enzymatic condensation reaction between the carbonyl group on reducing sugars (such as glucose) and the free amino group on macromolecules such as proteins, lipids or nucleic acids. The stable covalent adducts generated by the NEG reaction are collectively referred to as advanced glycation end products (AGEs). In the biochemical experiment, the glucose solution was mixed with the bovine serum albumin (BSA) solution, and aminoguanidine was used as the positive control and the phosphate buffer was used as the blank control. The mixed samples were reacted at 55°C in the dark for 4 days, and the inhibition rate was detected and calculated by a fluorescence microplate reader under the conditions of excitation wavelength of 370nm and emission wavelength of 440nm. The anti-glycation effect of the sample was judged by the inhibition rate.

[0094] 2. Test materials:

[0095] 2.1 Main reagents: bovine serum albumin (Sigma, M26HS122527), glucose (Sigma, SLBR0902V), PBS buffer (Boster, WH0621P201), aminoguanidine hydrochloride (Aladdin, G1909194).

[0096] 2.2 Main equipment: ELISA reader (BioTek, Epoch), vortex oscillator (Shanghai Qiqian Electronic Technology, MZX-2500), constant temperature incubator (Tianjin Test Instrument Co., Ltd., DH400BII), electronic balance (Sartorius, BSA124S-CW).

[0097] 3. Test groups and concentrations:

[0098]

[0099] 4. Test method:

[0100] A mixed solution containing 80 mg / mL bovine serum albumin and 240 mg / mL glucose was prepared using 0.1 mol / L phosphate buffer solution (PBS), filtered through a 0.22 μm filter membrane, and used as a 2× saccharification reaction solution. Each group of reaction systems was prepared according to the following table. The final concentration of bovine serum albumin in each group of reaction systems was 40 mg / mL, and the final concentration of glucose was 120 mg / mL.

[0101]

[0102] After mixing evenly, incubate at 55°C for 4 days. Use PBS or sample solvent instead of sample as negative control or solvent control, 1% aminoguanidine hydrochloride as positive control, and PBS instead of glycosylation reaction solution as control system. After the reaction is completed, cool the incubated solution to room temperature for determination, take 200 μL of the reaction solution and add it to a 96-well plate, and use a fluorescent microplate reader to detect and calculate the AGEs inhibition rate under the conditions of excitation wavelength 370 nm and emission wavelength 440 nm.

[0103] 5. Calculation of AGEs inhibition rate

[0104]

[0105] Where:

[0106] A- Fluorescence intensity of the saccharification system of the sample group

[0107] B- Fluorescence intensity of PBS solution of sample control group

[0108] C- Fluorescence intensity of the saccharification system in the blank control group

[0109] D- Fluorescence intensity of PBS solution in blank PBS group

[0110] 6. Test Results

[0111] Table 3

[0112]

[0113] The results showed that after artemisinic acid reacted with the glycosylation reaction solution in the mass concentration range of 0.0015 mg / mL to 0.0234 mg / mL, the AGEs inhibition rate was greater than 0% compared with the control group. It was able to significantly inhibit AGEs (P<0.01) and was dose-dependent, that is, the inhibition rate of artemisinic acid on AGEs increased with the increase of concentration, indicating that artemisinic acid has an anti-glycation effect.

[0114] Test Example 2: Artemisinic acid anti-glycated cell immunofluorescence experiment

[0115] 1. Test Principle

[0116] Carboxymethyl lysine (CML) is one of the representatives of advanced glycation end products. In the experiment, glyoxal was used to induce glycosylation in primary human skin fibroblasts, aminoguanidine was used as a positive control, and immunofluorescence staining was used to quantitatively analyze the expression of CML.

[0117] 2. Test Materials

[0118] 2.1 Main reagents: primary human dermal fibroblasts and culture medium ( Cell Technology), penicillin-streptomycin-amphotericin B solution (Bioind), DPBS (Gibco), CCK reagent (Tongren Biotechnology), CML antibody (Abcam), Alexa 568Donkey Anti-Mouse IgG (Invitrogen), bovine serum albumin BSA (Biyuntian), glyoxal (Adamas), aminoguanidine (Aladdin).

[0119] 2.2 Main equipment: cell culture incubator, inverted fluorescence microscope (Leica), biological safety cabinet, microplate reader.

[0120] 3. Test Methods

[0121] 3.1 Cell seeding:

[0122] After digesting the cells from the flask, inoculate them into a 24-well plate that has been added with cell slides, with 1.5*104 cells per well.

[0123] 3.2 Glycation induction:

[0124] After 48 hours, the cells were treated with 0.5 mM glyoxal to induce glycation reaction as a negative control group. At the same time, a blank group (NT), a control group (glyoxal) and a sample group (glyoxal + sample) were set up and the cells were treated for 48 hours.

[0125] 3.3 Fixation and staining:

[0126] The induced cells were rinsed once with PBS, fixed with paraformaldehyde (PFA), and then CML fluorescent staining was performed. After BSA blocking, primary antibody incubation and secondary antibody incubation, the cells were finally sealed with an aqueous mounting medium containing DAPI. Fluorescence images were taken with a fluorescence microscope, and the images were quantitatively analyzed using Image J.

[0127] 4. Test Results

[0128] Table 4

[0129]

[0130] The results showed that the glycation inhibition rates of Example 7 (0.00117 mg / mL artemisinic acid solution) and Example 8 (0.0059 mg / mL artemisinic acid solution) were 78.90% (P<0.001) and 33.31% (P<0.01), respectively, and there were significant differences. This indicates that artemisinic acid has a strong inhibitory effect on the glycation reaction of fibroblasts induced by glyoxal at a concentration of 0.00117 to 0.0059 mg / mL, has anti-glycation efficacy, and the effect of 0.00117 mg / mL concentration is better.

[0131] In addition, it can be seen from the figure that compared with the blank group ( Figure 2 ), control group ( Figure 3 ) After induction with glyoxal, the expression of advanced glycation end product CML in primary human dermal fibroblasts increased significantly, that is, the red CML staining area increased significantly. 0.00117mg / mL( Figure 4 ) and 0.0059 mg / mL( Figure 5 ) artemisinic acid solution was tested, the red area was significantly reduced compared with the control group, indicating that the expression of the advanced glycosylation end product CML in the cells was inhibited, and the inhibitory effect of 0.00117 mg / mL artemisinic acid solution was better than that of 0.0059 mg / mL artemisinic acid solution.

[0132] The artemisinic acid of the present invention can be used as an intermediate raw material or an effective additive for the preparation of medicines, health foods or skin topical preparations, and the skin topical preparations are preferably cosmetic compositions, including but not limited to the preparation of products in the form of creams, lotions, gels, lotions, essences, facial masks, eye creams, aerosols (cleansing bubbles), sprays, shower gels and facial cleansers. The artemisinic acid of the embodiment has a weight percentage of 0.0001%-20% (w / w) in the skin topical preparation. The preferred weight percentage is 0.001%-10% (w / w). The more preferred weight percentage is 0.001%-5% (w / w). The most preferred weight percentage is 0.01%-5% (w / w). In some embodiments, the weight percentage of artemisinic acid in the skin topical preparation is 0.00015%-0.00234% (w / w). The preferred weight percentages are 0.00059%, 0.00117%, and 0.00234% (w / w). More preferred weight percentages are 0.00117% and 0.00234% (w / w). The most preferred weight percentage is 0.00234% (w / w).

[0133] The following are examples of specific applications of artemisinic acid in skin external preparations, and the formulations and preparation methods of these preparations. In the following tables, "-" means no addition.

[0134] Application Example 1: Preparation of facial cream

[0135]

[0136]

[0137] Application Example 2: Preparation of Emulsion

[0138]

[0139]

[0140] Application Example 3: Preparation of Gel

[0141]

[0142] Application Example 4: Preparation of lotion

[0143]

[0144]

[0145] Application Example 5: Preparation of Essence

[0146]

[0147]

[0148] Application Example 6: Preparation of facial mask

[0149]

[0150] Application Example 7: Preparation of Eye Cream

[0151]

[0152]

[0153] Application Example 8: Preparation of Aerosol (Cleaning Bubble)

[0154]

[0155] Application Example 9: Preparation of Spray

[0156]

[0157]

[0158] Application Example 10: Preparation of shower gel

[0159]

[0160] Application Example 11: Preparation of facial cleanser

[0161]

Claims

1. Anti-glycation application of artemisinic acid.

2. The use according to claim 1, characterized in that The concentration of artemisinic acid used is at least 0.1 mg / L.

3. The use according to claim 2, characterized in that The concentration of artemisinic acid used is at least 1 mg / L.

4. The use according to claim 1, characterized in that The anti-glycation is the end product of inhibiting non-enzymatic glycosylation reaction.

5. The use according to claim 1, characterized in that The anti-glycation is to inhibit the glycation end products.

6. Application of artemisinic acid in the preparation of skin topical preparations, medicines and health foods with anti-glycation effects.

7. The use according to claim 6, characterized in that The content of artemisinic acid in skin external preparations, medicines and health foods is 0.00001-20% by weight.

8. The use according to claim 7, characterized in that The content of artemisinic acid in external skin preparations, medicines and health foods is 0.0001-5% by weight.

9. The use according to claim 6, 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.

10. The use according to claim 6, characterized in that The anti-glycation is the end product of inhibiting non-enzymatic glycosylation reaction.

11. The use according to claim 6, characterized in that The anti-glycation is to inhibit the glycation end products.

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