A Chinese medicine composition, extract, preparation method and application thereof, cosmetics and medicine

Abstract

A Chinese medicine composition, an extract, a preparation method and application thereof, cosmetics and medicines, belonging to the field of Chinese medicine. The Chinese medicine composition comprises: forsythia fruit, tribulus fruit, acanthopanax, polygonatum, immature bitter orange and ginseng. By using the mutual cooperation of specific raw materials, forsythia fruit, tribulus fruit, acanthopanax, polygonatum, immature bitter orange and ginseng, it has the ability to inhibit MMP-1 gene expression, promote TIMP-1 expression, inhibit the generation of reactive oxygen, promote the expression of NQO1 gene reduced by oxidative stress, inhibit NO generation and inhibit iNOS expression. Experiments show that it not only has antioxidant, anti-wrinkle and anti-inflammatory effects, but is also non-toxic, so it can be used in medicines, cosmetics and health products.

Description

Technical Field

[0001] The present application relates to the field of traditional Chinese medicine, and specifically, to a traditional Chinese medicine composition, an extract, a preparation method and application thereof, cosmetics and medicines. Background Art

[0002] Skin is a body organ that is in close contact with the external environment and has the function of protecting the internal organs of the human body. It can be roughly divided into three types: epidermis, dermis, and subcutaneous fat. Skin is a complex organ formed by cells with multiple functions and substances with matching physical properties. For the dermis, elastic solid substances (fibers) and viscous liquid substances are combined, so that the inherent skin elasticity can be maintained. Therefore, the skin responds to mechanical tension based on its unique physical properties, which is called viscoelasticity. The skin has this viscoelasticity because collagen fibers and elastin fibers form a unique three-dimensional structure in the matrix of proteoglycans. Generally, as we age, the skin's function declines and atrophic changes occur due to ultraviolet rays, pollution, pressure, etc., and the number of skin cells decreases or the thickness of the skin decreases. Due to this process, the elastic fibers of the skin are deformed, resulting in reduced skin elasticity or skin sagging. Collagen is the main component of the skin, of which type I collagen accounts for 80% and type III collagen accounts for 15%. When the skin is exposed to ultraviolet rays for a long time, a large amount of active oxygen is generated in the skin, thereby promoting the expression or activity of MMP-1 (matrix metalloproteinase-1), an enzyme that decomposes collagen among matrix metalloproteinases (MMPs), and reducing collagen synthesis, thereby causing a decrease in skin elasticity and the formation of wrinkles.

[0003] As one of the body's defense mechanisms against physical stimulation, chemical stimulation, bacterial infection, etc., the inflammatory response is one of the mechanisms used to remove external substances and repair and regenerate damaged parts. Monocytes are activated by bacterial infection, etc. and can be transformed into macrophages with phagocytic function. Macrophages secrete NO, prostaglandin E2, proinflammatory cytokines, etc., thereby inducing inflammation, and are used as important regulatory cells in inflammation and immune response. In order to achieve this process, macrophages need to undergo an activation process. Lipopolysaccharide (LPS), which is one of the cell wall structural components of Gram-negative bacteria and is well known as endotoxin, is the most famous external factor involved in the activation of macrophages. In particular, because monocytes or macrophages such as RAW 264.7 cells secrete proinflammatory cytokines such as TNF-α, IL-6, and IL-1β, LPS in the inflammatory site increases. When LPS stimulates macrophages, nitric oxide (NO) is generated during the process of converting L-arginine into L-citrulline by an enzyme called inducible nitric oxide synthase (iNOS), and thus macrophages produce NO.

[0004] NO in mammals is synthesized by three NO synthases (NOS), namely nNOS (neuronal nitric oxide synthase), eNOS (endothelial nitric oxide synthase) and iNOS. Among them, NO generated by nNOS and eNOS is generated for normal biological functions, and its concentration in the tissue is maintained at a low prescribed level. However, NO generated by iNOS can show pathological vasodilation, cytotoxicity, tissue damage and other harmful effects on the organism due to its excessive amount. In addition, prostaglandin E2 (PGE2) as an inflammatory factor is formed by arachidonic acid being catalyzed by cyclooxygenase (COXs), thereby inducing an inflammatory reaction, wherein the above-mentioned arachidonic acid is stimulated by LPS to make phospholipids as a structural component of the cell membrane free by phospholipase A2 (phospholipase A2). COX is divided into COX-1 and COX-2, COX-1 plays a role in normal biological functions such as platelet formation, gastric wall protection, and maintenance of renal function in the body, and COX-2 synthesizes PHE2 as an inflammatory mediator. It is known that PHE2 is closely related to the occurrence of cancer, such as promoting inflammatory responses (pain, fever, etc.), immune responses, and angiogenesis.

[0005] Nonsteroidal anti-inflammatory drugs (NSAIDs) including selective COX-2 inhibitors have anti-inflammatory, antipyretic and analgesic effects, however, long-term use of these drugs can sometimes lead to serious side effects, such as secondary anemia caused by peptic ulcer bleeding related to the gastrointestinal tract, inhibition of platelet function, inhibition of labor induction, side effects on the kidneys, liver damage, allergic reactions, etc. Summary of the invention

[0006] The present application provides a traditional Chinese medicine composition, an extract, a preparation method and application thereof, a cosmetic and a medicine, which can improve at least one of the above-mentioned technical problems.

[0007] The embodiment of the present application is implemented as follows:

[0008] In a first aspect, the present application provides a traditional Chinese medicine composition, which includes: Forsythia suspensa fruit, Tribulus terrestris fruit, Acanthopanax senticosus, Polygonatum odoratum, Citrus aurantium and Ginseng.

[0009] In the second aspect, the present application provides an extract, which is obtained by extracting the traditional Chinese medicine composition provided in the first aspect of the present application.

[0010] In the third aspect, the present application provides an example of a method for preparing the above-mentioned extract, which is obtained by extracting the traditional Chinese medicine composition provided in the first aspect of the present application with an extractant.

[0011] Wherein, the extractant is selected from water, C 1 -C 4 Any of alcohol-water solutions.

[0012] The Chinese medicine composition provided in the first aspect, the extract provided in the second aspect, and the extract extracted by the preparation method provided in the third aspect all utilize the cooperation of specific raw materials, including Forsythia suspensa fruit, Tribulus terrestris fruit, Acanthopanax senticosus, Polygonatum odoratum, Citrus aurantium and Ginseng, and have the ability to inhibit MMP-1 gene expression, promote TIMP-1 expression, inhibit reactive oxygen generation, promote NQO1 gene expression reduced by oxidative stress, inhibit NO generation and inhibit iNOS expression. Experiments have shown that it not only has antioxidant, anti-wrinkle and anti-inflammatory effects, but is also non-toxic, and can therefore be used in the fields of medicines, cosmetics, etc.

[0013] Therefore, in the fourth aspect, the present application provides an example of the use of the Chinese medicine composition provided in the first aspect, the extract provided in the second aspect, or the extract extracted by the preparation method provided in the third aspect in the preparation of a preparation for inhibiting the generation of reactive oxygen species and / or promoting the recovery of NQO1 gene expression.

[0014] In the fifth aspect, the present application provides an example of the use of the Chinese medicine composition provided in the first aspect, the extract provided in the second aspect, or the extract extracted by the preparation method provided in the third aspect in the preparation of a preparation for inhibiting NO production and / or inhibiting iNOS expression.

[0015] In the sixth aspect, the present application provides an example of the use of the Chinese medicine composition provided in the first aspect, the extract provided in the second aspect, or the extract extracted by the preparation method provided in the third aspect in the preparation of anti-inflammatory, antioxidant and / or anti-wrinkle preparations, wherein the preparations include any one of cosmetics and medicines.

[0016] In the seventh aspect, the present application provides an example of the use of the Chinese medicine composition provided in the first aspect, the extract provided in the second aspect, or the extract extracted by the preparation method provided in the third aspect in the preparation of a preparation for inhibiting MMP-1 gene expression and / or promoting TIMP-1 expression.

[0017] In an eighth aspect, the present application provides a cosmetic comprising the Chinese medicine composition provided in the first aspect, the extract provided in the second aspect, or the extract extracted by the preparation method provided in the third aspect.

[0018] In the ninth aspect, the present application provides an example of a medicine, which includes the Chinese medicine composition provided in the first aspect, the extract provided in the second aspect, or the extract extracted by the preparation method provided in the third aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 is the toxic effect of the extract on HaCaT cells;

[0021] Figure 2 is the effect of the extract on the expression of collagen;

[0022] Figure 3 Effects of the extract on the expression of MMP-1 gene;

[0023] Figure 4 Effects of extracts on TIMP-1 expression;

[0024] Figure 5 is the effect of the extract on reactive oxygen species produced due to oxidative stress;

[0025] Figure 6 is the effect of the extract on NQO1 gene expression;

[0026] Figure 7 is the effect of the extract on NO production and cytotoxicity to Raw264.7 cells;

[0027] Figure 8 is the effect of the extract on iNOS gene expression;

[0028] Fig. 9 is the effect of the extract on COX-2 gene expression. DETAILED DESCRIPTION

[0029] The embodiments of the present application will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. In the examples, if specific conditions are not specified, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0030] In the first aspect, the present application provides a Chinese medicine composition, which includes: forsythia fruit, tribulus fruit, acanthopanax, polygonatum, immature bitter orange and ginseng. Forsythia fruit, tribulus fruit, acanthopanax, polygonatum, immature bitter orange and ginseng cooperate with each other to enhance synergy, and have the ability to inhibit MMP-1 gene expression, promote TIMP-1 expression, inhibit reactive oxygen generation, promote NQO1 gene expression reduced by oxidative stress, inhibit NO generation and inhibit iNOS expression. It not only has anti-inflammatory, antioxidant and post-oxidation repair effects, but also has the ability to promote collagen production, etc., so as to resist wrinkles and skin aging, etc., so it is particularly suitable for cosmetics with various skin improvement effects.

[0031] Among them, forsythia fruit refers to the dried fruit of Forsythia viridissima Lindley or Forsythia suspensa Vahl of the Oleaceae family. The medicinal effect has the effects of reducing fever and detoxification, eliminating wind-heat, dissolving swelling of boils or wounds, or reducing swelling and dissolving knots; its main chemical components include forsythia phenol, sterols, saponins, oleanolic acid, etc.

[0032] Tribulus terrestris fruit refers to the fruit of Tribulus terrestris L. of the family Tribulaceae. The drug is pungent, bitter, and slightly warm in nature. It is used for dizziness, headache, hallucinations, hypertension, chest and flank pain, menstrual disorders, milk secretion, mastitis, eye congestion, white fur, eczema, urticaria, neurodermatitis, and pruritus caused by the rise of liver qi. As pharmacological effects, it has been reported to reduce blood pressure, diuresis, relieve cough, eliminate phlegm, and relieve asthma.

[0033] Eleutherococcus senticosus is used as a medicine for treating stroke or weak constitution in folk or traditional Chinese medicine. Eleutherococcus senticosus bark wine is very effective for low back pain, numbness of hands and feet, hemiplegia, etc.

[0034] Polygonatum odoratum var.pluriflorum is the rhizome of Polygonatum odoratum (Polygonatum odoratum var.pluriflorum), a plant of the Liliaceae family. Polygonatum odoratum has a tonic effect and is therefore used to treat heart failure, etc. It is also effective for irritability, dry mouth, and diabetes.

[0035] Ponciri Fructus Immaturus (KP) is the unripe fruit of the Rutaceae family, which is used for ventilation, stomachic, diuretic, expectorant and analgesia.

[0036] Ginseng is a perennial plant of the genus Panax. Specific examples of ginseng used in this application include: Korean ginseng (Panax ginseng), American ginseng (Panax quinquefolia), Panax notoginseng (Panax notoginseng), bamboo ginseng (Panax japonicus), Himalayan ginseng (Panaxa pseudoginseng), Vietnamese ginseng (Panax vietnamensis), beautiful ginseng (Panax elegatior), narrow-leaved bamboo ginseng (Panax wangianus), feather leaf Panax (Panax bipinratifidus), etc. Ginseng has a good effect on improving immunity, relieving fatigue, improving blood circulation, relieving menopause, improving memory, and has multiple anti-cancer effects.

[0037] Optionally, the Chinese medicine composition comprises: 100 parts by weight of Forsythia fruit, 100-150 parts by weight of Tribulus terrestris fruit, 75-125 parts by weight of Acanthopanax senticosus, 100-150 parts by weight of Polygonatum odoratum, 25-50 parts by weight of Citrus aurantium, and 50-75 parts by weight of Radix Ginseng. The specific range of the above raw materials is adopted, and the ratio is reasonable. Under the specific ratio provided in the present application, the composition can achieve a good synergistic effect.

[0038] It should be noted that the above-mentioned Chinese medicine composition can be used independently or in combination with other non-conflicting raw materials or auxiliary materials, and no limitation is made here.

[0039] In a second aspect, the present application provides an extract obtained by extracting the above-mentioned Chinese medicine composition. The extract has the ability to inhibit MMP-1 gene expression, promote TIMP-1 expression, inhibit reactive oxygen generation, promote NQO1 gene expression reduced by oxidative stress, inhibit NO generation, and inhibit iNOS expression.

[0040] In a third aspect, the present application provides a method for preparing the above-mentioned extract, which is obtained by extracting the above-mentioned traditional Chinese medicine composition with an extractant.

[0041] Wherein, the extractant is selected from water, C 1 -C 4 At least one of alcohols; illustratively, C 1 -C 4 The alcohol may be selected from at least one of methanol, ethanol, propanol, isopropanol, butanol and isobutanol.

[0042] Optionally, the extractant is an aqueous ethanol solution.

[0043] Optionally, the volume fraction of ethanol in the ethanol aqueous solution is 25-35%, for example, the volume fraction of ethanol in the ethanol aqueous solution is 25%, 30%, 33% or 35%, etc.

[0044] Exemplarily, the extraction includes: immersing the raw material to be extracted in an extractant for extraction, wherein the amount of the extractant added is 1 to 40 times the weight of the raw material to be extracted, specifically 5 to 40 times, 10 to 30 times, etc., which can be limited by those skilled in the art according to actual needs. The extracted raw material can be a traditional Chinese medicine composition or each raw material of the traditional Chinese medicine composition.

[0045] That is, the raw materials may be mixed according to a ratio and then extracted together, or the raw materials may be extracted independently after being mixed according to a ratio and then mixed, which is not limited here.

[0046] In the actual extraction process, the raw material to be extracted can be directly immersed in the extractant for a period of time for extraction. There is no special restriction on the extraction time, which can be selected according to actual needs. In order to avoid waste due to too short extraction time and the influence of some components due to too long extraction time, the extraction time can be within 10 minutes to 2 days.

[0047] In order to speed up the extraction process, heating, ultrasonic extraction, etc. can be assisted in the extraction process. Optionally, in some optional examples shown in the present application, the extraction includes: ultrasonic extraction for 1.5-3h, such as 1.5h, 1.7h, 2h, 2.5h, 2.7h or 3h, etc.

[0048] Optionally, the extraction further includes concentration, and after concentration, it can be dried according to actual needs.

[0049] The equipment used for the above extraction can be selected from conventional extraction equipment, ultrasonic crushing extractor or grader according to actual needs. The extract prepared in this way can be removed from the extractant by hot air drying, reduced pressure drying or freeze drying, which is convenient and quick.

[0050] It should be noted that, in addition to the above-mentioned extraction methods, conventional methods such as immersion combined chromatography can also be used alone or in appropriate combination to fractionate or purify the extracts by using known methods for separating and extracting plant components, which will not be described in detail here. Among them, the chromatography method can be selected from any one of silica gel column chromatography, LH-20 column chromatography, ion exchange resin chromatography, medium pressure liquid chromatography, thin layer chromatography, silica gel vacuum liquid chromatography and high performance liquid chromatography.

[0051] Based on the effects of the above-mentioned Chinese medicine composition and extract, the present application provides specific applications of the above-mentioned Chinese medicine composition, the above-mentioned extract, or the extract prepared by the above-mentioned preparation method.

[0052] Specifically, in the third aspect, the present application provides the use of the Chinese medicine composition provided in the first aspect, the extract provided in the second aspect, or the extract extracted by the preparation method provided in the third aspect in the preparation of a preparation for inhibiting the generation of reactive oxygen species and / or promoting NQO1 gene expression.

[0053] Among them, inhibiting the generation of reactive oxygen species and / or promoting the expression of the NQO1 gene is not only related to the antioxidant effect, but also external conditions such as ultraviolet rays and blue light can induce oxidative stress in the skin, causing certain damage to the skin, resulting in reduced skin elasticity and the formation of wrinkles. Therefore, the above-mentioned preparations that inhibit the generation of reactive oxygen species and / or promote the expression of the NQO1 gene will also have certain repair and anti-aging capabilities for damaged skin.

[0054] In a fourth aspect, the present application provides the use of the Chinese medicine composition provided in the first aspect, the extract provided in the second aspect, or the extract extracted by the preparation method provided in the third aspect in preparing a preparation for inhibiting NO production and / or inhibiting iNOS expression.

[0055] Among them, the preparations that inhibit NO generation and / or inhibit iNOS expression mainly have anti-inflammatory effects.

[0056] In the fifth aspect, the present application provides the use of the Chinese medicine composition provided in the first aspect, the extract provided in the second aspect, or the extract extracted by the preparation method provided in the third aspect in the preparation of a preparation for inhibiting MMP-1 gene expression and / or promoting TIMP-1 expression.

[0057] Among them, inhibiting MMP-1 gene expression and / or promoting TIMP-1 expression are related to the synthesis of collagen, and preparations that inhibit MMP-1 gene expression and / or promote TIMP-1 expression can effectively resist wrinkles and anti-aging.

[0058] It should be noted that the above-mentioned preparations can be medicines, cosmetics, health products or experimental intermediate reagents, which are not limited here.

[0059] In summary, based on the effects of the above-mentioned Chinese medicine composition, the above-mentioned extract, or the extract prepared by the above-mentioned preparation method, in the sixth aspect, the present application provides the use of the Chinese medicine composition provided in the first aspect, the extract provided in the second aspect, or the extract extracted by the preparation method provided in the third aspect in the preparation of anti-inflammatory, antioxidant and / or anti-wrinkle preparations, and the preparations include any one of cosmetics and medicines.

[0060] It should be noted that, in the present application, anti-inflammatory means that the preparation can effectively improve or treat inflammatory diseases.

[0061] Inflammatory diseases include at least one of inflammatory bowel disease, inflammatory collagen vascular disease, glomerulonephritis, sarcoidosis, atopic dermatitis, retinitis, gastritis, hepatitis, enteritis, arthritis, tonsillitis, pharyngitis, bronchitis, pneumonia, pancreatitis, cystitis, neuritis, sepsis and nephritis.

[0062] In a seventh aspect, the present application provides a cosmetic comprising the Chinese medicine composition provided in the first aspect, the extract provided in the second aspect, or the extract extracted by the preparation method provided in the third aspect.

[0063] Among them, this cosmetic has multiple effects such as anti-inflammation on skin cells, improvement of wrinkles, anti-oxidation, and anti-aging.

[0064] Optionally, the amount of the extract added to the cosmetic is, for example, 0.01 to 5 wt%, specifically, 0.01 wt%, 0.1 wt%, 0.5 wt%, 1 wt%, 1.3 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt% or 5 wt%, etc. Optionally, the amount of the extract added to the cosmetic is 0.01 to 3 wt%. It should be noted that the extract herein is at least one of the extract provided in the second aspect or the extract extracted by the preparation method provided in the third aspect.

[0065] Cosmetics can specifically include essence, lotion, emulsion, facial mask, hand cream, foot cream, lip balm, lipstick, eye shadow, eyeliner, eyebrow pencil, blush, highlighter, ordinary lotion, toner, cream, essence, beauty soap, soft lotion, medicinal lotion, body cleanser, cleansing foam, cleansing milk, gel, makeup remover oil, cleansing cream, shampoo, conditioner, hair oil, conditioner, makeup remover wipes or makeup remover water, etc.

[0066] That is, the dosage form of the cosmetic is any one of a paste, cream, gel, powder, spray, solution, emulsion, suspension, and a detergent containing a surfactant, etc. The dosage form of the cosmetic can be prepared in any dosage form commonly prepared in the art.

[0067] Among them, the cosmetics contain carrier ingredients that can be conventionally incorporated into cosmetics.

[0068] Specifically, when the cosmetic is in the form of a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicon dioxide, talc or zinc oxide can be used as a carrier component.

[0069] When the cosmetic is in the form of a powder or spray, lactose, talc, silicon dioxide, aluminum hydroxide, calcium silicate or polyamide powder can be used as a carrier component. In particular, when it is a spray, it can also include a propellant such as chlorofluorocarbons, propane / butane or dimethyl ether.

[0070] When the dosage form of the cosmetic is a solution or an emulsion, a solvent, a solvating agent or an emulsifier, for example, water, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol oil, aliphatic glycerol esters, polyethylene glycol or fatty acid esters of sorbitol can be used as a carrier component.

[0071] When the dosage form of the cosmetic is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan esters and polyoxyethylene sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth gum can be used as carrier components.

[0072] When the dosage form of the cosmetic is a cleanser containing a surfactant, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinic acid monoesters, isethionates, imidazoline-type surfactants, methyl taurates, sarcosinates, fatty acid amide ether sulfates, alkylamido betaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glyceryl fatty acid esters can be used as carrier components.

[0073] In addition to the above-mentioned raw materials, cosmetics may also contain auxiliary ingredients with certain skin care effects, as well as cosmetic additives that can be conventionally incorporated into cosmetics according to actual dosage form requirements.

[0074] Specifically, the auxiliary ingredients include but are not limited to at least one of water-soluble vitamins and their salts or derivatives, oil-soluble vitamins and their derivatives, high-molecular polypeptides, high-molecular polysaccharides, sphingolipids and seaweed extracts.

[0075] The water-soluble vitamins herein may specifically include at least one of vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, niacin, niacinamide, folic acid, vitamin C, and vitamin H, etc., the water-soluble vitamin salts may include thiamine hydrochloride or sodium ascorbate, etc., the water-soluble vitamin derivatives may include at least one of sodium ascorbate-2-phosphate, magnesium ascorbate-2-phosphate, etc. The water-soluble vitamins and their salts or derivatives may be obtained by conventional methods such as microbial transformation, purification from microbial cultures, enzymatic methods, or chemical synthesis methods.

[0076] Oil-soluble vitamins may specifically include vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (DL-α-tocopherol, D-α-tocopherol, D-α-tocopherol), etc., and derivatives of oil-soluble vitamins may include at least one of ascorbyl palmitate, ascorbyl stearate, ascorbyl dipalmitate, acetic acid dl-α-tocopherol, nicotinic acid dl-α-tocopherol vitamin E, DL-panthenol, D-panthenol, pantothenic acid alcohol ethyl ether, etc. Oil-soluble vitamins may be obtained by conventional methods such as microbial transformation, purification from microbial cultures, enzymatic methods, or chemical synthesis methods.

[0077] The high molecular weight polypeptide may include at least one of collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, keratin, etc. The high molecular weight polypeptide may be purified and obtained by conventional methods such as purification from microbial culture, enzymatic method or chemical synthesis, or may be purified and used from natural products such as dermis of pigs or cattle, silk fibers of silkworms, etc.

[0078] The high molecular weight polysaccharide may include at least one of hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate or its salt (sodium salt, etc.), etc. Among them, chondroitin sulfate or its salt, etc. can be purified from conventional mammals or fishes for use.

[0079] Sphingolipids may specifically include at least one of ceramide, phytosphingosine, sphingolipids, etc. Sphingolipids can usually be purified from mammals, fish, shellfish, yeast, plants, etc. by conventional methods or obtained by chemical synthesis.

[0080] The seaweed extract may include at least one of brown algae extract, red algae extract, green algae extract, etc. In addition, the seaweed extract used in the present application also includes carrageenan, alginic acid, sodium alginate, potassium alginate, etc. purified from the seaweed extract. The seaweed extract can be obtained by purifying from seaweed by conventional methods.

[0081] Cosmetic additives may include at least one of oil components, moisturizers, softeners, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohol, pigments, fragrances, blood circulation promoters, coolants, antiperspirants and purified water. The specific selection can be based on the actual dosage form requirements.

[0082] Among them, the oil component may include ester oils, hydrocarbon oils, silicone oils, fluorine oils, animal oils, food oils and the like.

[0083] The ester oil can be tri-2-ethylhexanoin, hexadecyl 2-ethylhexanoate, isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate, isocetyl isostearate, butyl stearate, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl myristate, isostearyl myristate, isostearyl palmitate, octyldodecyl myristate, isocetyl isostearate, diethyl sebacate, diisopropyl adipate, isoalkyl neopentanoate, tri(caprylic acid, capric acid)glyceryl, tri-2-ethylhexanoin trimethylolpropane ester, triisostearate trimethylolpropane ester, tetra Pentaerythritol 2-ethylhexanoate, cetyl octanoate, decyl laurate, hexyl laurate, decyl myristate, myristyl myristate, cetyl myristate, stearyl stearate, decyl oleate, cetyl ricinoleate, isostearyl laurate, isotridecyl myristate, isocetyl palmitate, octyl stearate, isocetyl stearate, isodecyl oleate, octyldodecyl oleate, octyldodecyl linoleate, isopropyl isostearate, cetearyl 2-ethylhexanoate, stearyl 2-ethylhexanoate, hexyl isostearate, ethylene glycol dicaprylate, ethylene glycol dioleate, propylene glycol dicaprate, di(octanoic acid, Propylene glycol decanoate, propylene glycol dicaprylate, neopentyl glycol dicaprate, neopentyl glycol dicaprylate, tricaprylyl, trihexadecanoic acid, glyceryl triisopalmitate, glyceryl triisostearate, octyldodecyl neopentanoate, isostearyl octanoate, octyl isononanoate, hexyldecyl neodecanoate, octyldodecyl neodecanoate, isocetyl isostearate, isostearyl isostearate, octyldecyl isostearate, polyglyceryl oleate, polyglyceryl isostearate, triisocetyl citrate, triisoalkyl citrate, triisooctyl citrate, lauryl lactate, myristyl lactate, cetyl lactate, octyldecyl lactate, triethyl citrate The invention can be selected from the group consisting of esters, acetyl triethyl citrate, acetyl tributyl citrate, trioctyl citrate, diisostearyl malate, 2-ethylhexyl hydroxystearate, di-2-ethylhexyl succinate, diisobutyl adipate, diisopropyl sebacate, dioctyl sebacate, cholesterol stearate, cholesterol isostearate, cholesterol hydroxystearate, cholesterol oleate, dihydrocholesterol oleate, phytosteryl isostearate, phytosteryl oleate, isocetyl 12-stearoyl hydroxystearate, stearyl 12-stearoyl hydroxystearate, or isostearyl 12-stearoyl hydroxystearate.

[0084] The hydrocarbon oil may include one or more of squalene, liquid paraffin, α-olefin oligomers, isoparaffin, ozokerite, paraffin, liquid isoparaffin, polybutene, microcrystalline wax, vaseline and the like.

[0085] Silicone oils and fats may include one or more of polymethyl silicone, methylphenyl silicone, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane·methylcetyloxysiloxane copolymer, dimethylsiloxane·methylstearyloxysiloxane copolymer, alkyl-modified silicone oil and amino-modified silicone oil.

[0086] Fluorine-based greases may include perfluoropolyethers.

[0087] Animal or food oils and fats may include one or more of avocado oil, almond oil, olive oil, sesame oil, rice bran oil, safflower oil, soybean oil, corn oil, rapeseed oil, almond oil, palm kernel oil, palm oil, castor oil, sunflower oil, grapeseed oil, cottonseed oil, coconut oil, macadamia oil, wheat germ oil, rice germ oil, shea butter, evening primrose oil, macadamia nut oil, meadowfoam seed oil, egg yolk oil, butter, horse oil, mink oil, deep sea fish oil, jojoba oil, candelilla wax, carnauba wax, liquid lanolin, hydrogenated castor oil, etc.

[0088] The moisturizer may include a water-soluble low-molecular moisturizer, a fat-soluble low-molecular moisturizer, a water-soluble high-molecular moisturizer or a fat-soluble high-molecular moisturizer.

[0089] Among them, water-soluble low-molecular moisturizers may include serine, glutamine, sorbitol, mannitol, sodium pyrrolidone-carboxylate, glycerol, propylene glycol, 1,3-butylene glycol, ethylene glycol, polyethylene glycol B (polymerization degree n=2 or more), polypropylene glycol (polymerization degree n=2 or more), polyglycerol B (polymerization degree n=2 or more), lactic acid, lactate, etc.

[0090] Fat-soluble low molecular weight moisturizers may include cholesterol, cholesterol esters, and the like.

[0091] The water-soluble polymer may include carboxyvinyl polymer, polyaspartate, tragacanth gum, xanthan gum, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, water-soluble chitin, chitosan, dextrin and the like.

[0092] The fat-soluble polymer may include polyvinyl pyrrolidone-eicosene copolymer, polyvinyl pyrrolidone-hexadecene copolymer, nitrocellulose, dextrin fatty acid ester, polymer silicone and the like.

[0093] The softening agent may include long chain acyl glutamic acid cholesterol ester, hydroxystearate cholesterol ester, 12-hydroxystearic acid, stearic acid, rosin acid, lanolin fatty acid cholesterol ester and the like.

[0094] Among them, the surfactant may include nonionic surfactant, anionic surfactant, cationic surfactant, amphoteric surfactant and the like.

[0095] Specifically, the nonionic surfactant may include self-emulsifying monostearate glyceryl, propylene glycol fatty acid ester, glycerol fatty acid ester, polyglycerol fatty acid ester, sorbitan fatty acid ester, POE (polyoxyethylene) sorbitan fatty acid ester, POE sorbitol fatty acid ester, POE glycerol fatty acid ester, POE alkyl ether, POE fatty acid ester, POE hydrogenated castor oil, POE castor oil, POE·POP (polyoxyethylene·polyoxypropylene) copolymer, POE·POP alkyl ether, polyether modified silicone, lauric acid alkanolamide, alkylamine oxide, hydrogenated soybean lecithin, etc.

[0096] Anionic surfactants may include fatty acid soaps, α-acyl sulfonates, alkyl sulfonates, alkyl allyl sulfonates, alkyl naphthalene sulfonates, alkyl sulfates, POE alkyl ether sulfates, alkyl amide sulfates, alkyl phosphates, POE alkyl phosphates, alkyl amide phosphates, alkanoyl alkyl taurates, N-acyl amino acid salts, POE alkyl ether carboxylates, alkyl sulfosuccinates, sodium alkyl sulfoacetates, acylated hydrolyzed collagen peptide salts, perfluoroalkyl phosphates, and the like.

[0097] Cationic surfactants may include alkyltrimethylammonium chloride, stearyltrimethylammonium chloride, stearyltrimethylammonium bromide, cetearyltrimethylammonium chloride, distearyldimethylammonium chloride, stearyldimethylbenzyl ammonium chloride, behenyltrimethylammonium bromide, benzalkonium chloride, diethylaminoethylamide stearate, dimethylaminopropylamide stearate, quaternary ammonium salts of lanolin derivatives, and the like.

[0098] The amphoteric surfactant may include carboxybetaine type, amide betaine type, sulfobetaine type, hydroxysulfobetaine type, amide sulfobetaine type, phosphate betaine type, aminocarboxylate type, imidazoline derivative type, amide amine type and the like.

[0099] Inorganic pigments may include, for example, silicic acid, anhydrous silicic acid, magnesium silicate, talc, sericite, mica, kaolin, ferric oxide, clay, bentonite, titanium-coated mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, aluminum oxide, calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, ultramarine, chromium oxide, chromium hydroxide, calamine, and complexes thereof.

[0100] Organic pigments may include polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenolic resin, fluororesin, silicone resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, divinylbenzene styrene copolymer, silk powder, cellulose, CI pigment yellow, CI pigment orange, etc.

[0101] The organic powder may include metal soaps such as calcium stearate; alkyl phosphate metal salts such as sodium zinc cetyl phosphate, zinc lauryl phosphate, calcium lauryl phosphate, etc.; acyl amino acid polyvalent metal salts such as calcium N-lauroyl-β-alanine, zinc N-lauroyl-β-alanine, calcium N-lauroyl glycinate, etc.; amide sulfonic acid polyvalent metal salts such as calcium N-lauroyl taurate, calcium N-palmitoyl taurate, etc.; N-acyl basic amino acids such as N-ε-lauroyl-L-lysine, N-ε-palmitoyl lysine, N-α-palmitoyl ornithine, N-α-lauroyl arginine, N-α-hydrogenated tallow fatty acid acyl arginine, etc.; N-acyl polypeptides such as N-lauroyl glycylglycine, etc.; α-amino fatty acids such as α-amino caprylic acid, α-amino lauric acid, etc.; polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, divinylbenzene·styrene copolymer, tetrafluoroethylene, etc.

[0102] The ultraviolet absorber may include p-aminobenzoic acid, ethyl p-aminobenzoate, amyl p-aminobenzoate, octyl p-aminobenzoate, glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, trimethylol salicylate, benzyl cinnamate, 2-ethoxyethyl p-methoxycinnamate, octyl p-methoxycinnamate, mono-2-ethylhexyl dimethoxycinnamate, isopropyl p-methoxycinnamate, diisopropyl diisopropyl cinnamate, and the like. mixture, urocanic acid, ethyl urocanate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenone sulfonic acid and their salts, dihydroxymethoxybenzophenone, dihydroxymethoxybenzophenone sodium disulfonate, dihydroxybenzophenone, tetrahydroxybenzophenone, 4-tert-butyl-4'-methoxydibenzoylmethane, 2,4,6-triphenylamino-p-(carbonyl-2'-ethylhexyl-1'-oxy)-1,3,5-triazine, 2-(2-hydroxy-5-methylphenyl)benzotriazole, etc.

[0103] Bactericides may include cypress phenol, triclosan, trichlorohydroxydiphenyl ether, chlorhexidine gluconate, phenoxyethanol, resorcinol, isopropyl methylphenol, azulene, salicylic acid, zinc pyrithione, benzalkonium chloride, photosensitizer 301, sodium mononitroguaiaretic acid, undecylenic acid, and the like.

[0104] The antioxidants here are mainly used to prevent the product from oxidative deterioration. The antioxidants here may include butylated hydroxyanisole, propyl gallate, sorbic acid, and the like.

[0105] The pH adjuster may include citric acid, sodium citrate, malic acid, sodium malate, fumaric acid, sodium fumarate, succinic acid, sodium succinate, sodium hydroxide, disodium hydrogen phosphate, and the like.

[0106] The alcohol may include higher alcohols such as cetyl alcohol.

[0107] In addition, the mixed components that can be added are not limited thereto, and any one of the above-mentioned components can be mixed within the range that does not impair the purpose and effect of the present application, and no limitation is made here.

[0108] In a ninth aspect, the present application provides an example of a medicine, which includes the Chinese medicine composition provided in the first aspect, the extract provided in the second aspect, or the extract extracted by the preparation method provided in the third aspect.

[0109] Optionally, the amount of the extract added to the medicine is, for example, 0.001-30wt%, specifically, for example, the amount of the extract added to the medicine is 0.001wt%, 0.01wt%, 0.1wt%, 1wt%, 3wt%, 5wt%, 8wt%, 10wt%, 15wt%, 17wt%, 20wt%, 23wt%, 25wt%, 28wt% or 30wt%, etc. It should be noted that the extract here is at least one of the extracts provided in the second aspect or the extracts extracted by the preparation method provided in the third aspect.

[0110] In the case of preparing a formulation, a filler, an extender, a binder, a wetting agent, a diluent or an excipient such as a disintegrant and a surfactant are usually used for preparation.

[0111] Therefore, in addition to the Chinese medicine composition provided in the first aspect, the extract provided in the second aspect, or the extract extracted by the preparation method provided in the third aspect, the medicine may also include carriers, excipients and diluents according to actual dosage form requirements, and specific examples may include: lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0112] The drug may be in the form of an oral dosage form or a non-oral dosage form.

[0113] Among them, oral dosage forms include solid preparations such as powders, granules, pills, tablets, capsules, etc., which can be prepared by mixing the extract of the present application with at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

[0114] Oral dosage forms include liquid preparations such as suspensions, emulsions, syrups or aerosols, which may include various excipients such as wetting agents, sweeteners, aromatics and preservatives in addition to simple diluents such as commonly used water and liquid paraffin.

[0115] Non-oral preparations include external preparations, suppositories or injections.

[0116] As the base of the suppository, semi-synthetic fatty acid glyceride, polyethylene glycol, Tween 61, cocoa butter, lauric acid glyceride, glycerin gelatin and the like can be used.

[0117] The external preparation or injection specifically includes sterile aqueous solution, non-aqueous solvent, suspension, emulsion, lyophilized preparation, etc. As non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate can be used as diluent.

[0118] The dosage of the above-mentioned medicine is different according to the age, sex, body weight, specific disease to be treated or pathological condition, severity of disease or pathological condition, route of administration and the judgment of the prescriber of the object to be treated. The determination of the dosage based on these factors is within the cognitive level of those skilled in the art, and the dosage is usually in the range of 0.01mg / kg / day to about 2000mg / kg / day. A preferred dosage is 1mg / kg / day to 500mg / kg / day. Administration can be once a day or divided into several times. The above-mentioned dosage does not limit the scope of the application in any way.

[0119] The drug of the present application can be administered to mammals such as mice, livestock and humans by various routes, and all administration methods are contemplated, such as oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or intracerebral injection. Since the extract of the present application has almost no toxicity and side effects, the drug can be safely used even if taken for a long time for preventive purposes.

[0120] In addition, the Chinese medicine composition provided by the first aspect, the extract provided by the second aspect, or the extract extracted by the preparation method provided by the third aspect can be used in health functional foods, wherein the extract can be added to health functional foods at 0.001 weight percent to less than 100 weight percent.

[0121] The above-mentioned health functional foods include tablets, capsules, pills or liquid forms, and the extracts of the present application can be added to foods such as meat, sausages, bread, candies, snacks, noodles, ice cream, dairy products, soups, beverages, chewing gum, tea and vitamin complexes.

[0122] The following is a further detailed description of a Chinese medicine composition, extract, preparation method and application thereof, cosmetics and medicine of the present application in conjunction with the examples. In the following examples, the ginseng used is Korean ginseng.

[0123] Preparation of Extracts of Examples 1-7 and Comparative Examples 1-7

[0124] Extraction method: The Chinese medicine compositions shown in Examples 1-7 are configured according to Table 1, and the Chinese medicine compositions shown in Comparative Examples 1-7 are configured according to Table 2. After each Chinese medicine composition is immersed in a 30% (v / v) ethanol aqueous solution which is 10 times its weight, it is extracted by ultrasonic treatment at room temperature (25°C) (frequency is above 20 kHz, and extraction takes 2 hours). Subsequently, after concentrating with a rotary evaporator, butanediol is added to prepare a 30% (v / v) butanediol aqueous solution, and then diluted with a 30% (v / v) butanediol aqueous solution to obtain an extract solution with a solid content of 1% (w / w).

[0125] The obtained extract solution was used as a raw material (here, for ease of understanding, the extract solution is referred to as raw material A), and A was added to the cell culture medium at a concentration of 0.01 to 3% (w / w) and applied to the following test examples.

[0126] Table 1 Ratios of the Chinese medicine compositions shown in Examples 1-7

[0127]

[0128] Table 2 Ratios of Chinese medicine compositions shown in Comparative Examples 1-7

[0129]

[0130] Example 8

[0131] The 1% (w / w) solid content extract solution obtained in Example 1 was diluted in water to an aqueous extract solution having a solid content of 2.0% (w / w), and a lotion was prepared according to the composition of Table 3 below by the following preparation method.

[0132] The preparation method comprises: adding glycerin, butylene glycol, propylene glycol and a preservative to pure water in sequence, stirring and dissolving them, adding polyoxyethylene hydrogenated castor oil heated to about 60°C, adding fragrance and dissolving them, and then further heating the mixed solution to 60°C, adding the above-mentioned 2.0% (w / w) aqueous solution of the extract, ethanol, triethanolamine and pigment, stirring them thoroughly, and then ripening.

[0133] Table 3 Raw material composition and proportion of lotion

[0134] raw material Weight Percent Aqueous solution of extract with a solid content of 2% (w / w) 2.0 glycerin 3.0 Butanediol 2.0 Propylene glycol 2.0 Polyoxyethylene Hydrogenated Castor Seed Oil 1.0 Ethanol 10.0 Triethanolamine 0.1 Phenoxyethanol 0.4 Blue No. 1 0.01 spices 0.1 Purified water Up to 100

[0135] Example 9

[0136] The aqueous extract solution in Example 1 was diluted in water to an aqueous extract solution having a solid content of 2.0% (w / w), and a skin care lotion was prepared according to the composition of Table 4 below by the following preparation method.

[0137] The preparation method comprises: mixing and stirring propylene glycol, carboxyl polymer, preservative, and purified water, heating to 80-85°C, adding the mixture to the production department, and operating the emulsifier, heating beeswax, polysorbate 60, sorbitan stearate, liquid paraffin, sorbitan stearate, lipophilic monostearate glyceryl, stearic acid, glyceryl stearate / PEG-400 stearate, and triethanolamine to 80-85°C to dissolve the mixture, and then putting the mixture into the production department for emulsification. After the emulsification is completed, stirring and cooling to 50°C with a stirrer, adding flavors, cooling to 45°C, adding pigments, cooling to 35°C, adding the above-mentioned extract aqueous solution with a solid content of 2% (w / w), and finally cooling to 25°C for aging.

[0138] Table 4 Composition and proportion of skin care lotion raw materials

[0139] raw material Weight Percent Aqueous solution of extract with a solid content of 2% (w / w) 2.0 beeswax 1.0 Polysorbate 60 1.5 Sorbitan Stearate 0.5 Liquid paraffin 10.0 Sorbitan Stearate 1.0 Lipophilic monostearate glyceryl 0.5 Stearic acid 1.5 Glyceryl Stearate / PEG-400 Stearate 1.0 Propylene glycol 3.0 Carboxyl polymer 0.1 Triethanolamine 0.2 Phenoxyethanol 0.4 Blue No. 1 0.01 spices 0.1 Purified water Up to 100

[0140] Example 10

[0141] The extract obtained by concentration and freeze-drying in Example 1 was dissolved in water at 2.0% (w / w), and used as a raw material to prepare a cream according to the composition of Table 5 below by the following preparation method.

[0142] The preparation method comprises: mixing and stirring carboxyvinyl polymer, butylene glycol, glycerin and purified water, heating to 80-85°C, adding to the production section, operating the emulsifier, heating stearic acid, cetyl alcohol, glyceryl monostearate, polyoxyethylene sorbitan monostearate, sorbitan sesquioleate, glyceryl monostearate / glyceryl stearate / polyoxyethylene stearate, wax, liquid paraffin, squalane, caprylic / capric triglyceride to 80-85°C to dissolve them, adding triethanolamine and stirring, adding to the production section and emulsifying. After the emulsification is completed, stirring with a stirrer and cooling to 35°C, adding the above-mentioned 2% (w / w) extract aqueous solution, cooling to 25°C, and ripening.

[0143] Table 5 Cream raw material composition and ratio

[0144] raw material Content (g) 2% (w / w) extract in aqueous solution 2.0 Stearic acid 2.0 Cetyl Alcohol 2.0 Glyceryl Monostearate 2.0 Polyoxyethylene sorbitan monostearate 0.5 Sorbitan sesquioleate 0.5 Glyceryl Monostearate / Glyceryl Stearate / Polyoxyethylene Stearate 1.0 wax 1.0 Liquid paraffin 4.0 Squalane 3.0 Caprylic / capric triglyceride 6.0 Carboxyvinyl polymer 0.3 Butanediol 5.0 glycerin 3.0 Phenoxyethanol 0.4 Purified water Up to 100

[0145] It should be noted that in the following test examples, the concentration of the extract solution added refers to the concentration of the extract solution with a solid content of 1% (w / w) obtained in the above Examples 1-7 or Comparative Examples 1-7 as a whole added to the culture medium.

[0146] Test Example 1

[0147] The cell viability was determined by MTT colorimetry to evaluate the intracellular toxicity of each sample.

[0148] The principle of MTT colorimetry is as follows: MTT colorimetry is widely used as an experimental method for cell proliferation and toxicity by measuring the number of viable cells. For living cells, water-soluble MTT in the mitochondria as a yellow salt can be reduced to a water-insoluble blue formazan derivative by succinate dehydrogenase. The generated formazan derivative is usually dissolved by adding dimethyl sulfoxide (DMSO), and then the absorbance is measured.

[0149] Experimental method: HaCaT (human keratinocyte cell line) was cultured in DMEM medium containing 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) antibiotics at 37°C and 5% CO 2 cultured under the conditions of .

[0150] HaCaT cells in culture were cultured at 1.5×10 5 Cells / well were inoculated in a 24-well plate, 1 ml per well, and cultured for 24 hours. The culture medium in each well was replaced with a culture medium without fetal bovine serum containing the samples shown in Table 6, and then after culturing for 24 hours, the culture medium was removed, treated with 0.25 mg / ml MTT solution, and reacted at 37°C for 4 hours. After that, 1 ml of DMSO was added to the cells from which the MTT solution was removed to dissolve the MTT formazan, the absorbance was measured at 570 nm, and the cell survival rate was calculated according to the following mathematical formula.

[0151] Cell survival rate (%) = (sample absorbance value / average absorbance value of control group) × 100.

[0152] The results of cell survival rate are shown in Table 6 and Figure 1 .

[0153] Table 6 Cytotoxicity results on HaCaT

[0154]

[0155]

[0156] According to Table 6 and Figure 1 As shown in the results, compared with the control group, the extracts of Examples 1-7 provided in the present application had no cytotoxicity under the above-mentioned addition concentration conditions.

[0157] That is, the extract has no cytotoxicity when used in cosmetics or medicines.

[0158] Test Example 2

[0159] Increased effect on collagen synthesis in dermal fibroblasts

[0160] 1. Cultivation of Human Skin Fibroblasts

[0161] Human dermal fibroblasts were purchased from Promocell. Human dermal fibroblasts were cultured in DMEM medium (Gibco) at 37°C. The culture medium was maintained until the attached cells reached 80% confluence for subculture, during which the culture medium was replaced every two days until confluence. Subculture was performed as follows: the culture flask from which the culture medium was removed was washed with phosphate buffered saline (PBS), and the cells were detached with 0.25% trypsin-EDTA (GibcoBRL, Grand Island, NY, USA), and the cell suspension was centrifuged, the cell number was measured, and subculture was performed 3 times through the culture medium.

[0162] 2. Increased effect on collagen synthesis in dermal fibroblasts

[0163] In order to confirm the effect on the amount of collagen synthesis in dermal fibroblasts, a collagen measurement experiment was performed.

[0164] The subcultured human skin fibroblasts were added to DMEM medium supplemented with 10% (v / v) fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin, and incubated at 37°C with 5% CO 2 After 1 day of culture, the culture medium was removed and rinsed with PBS. The culture medium containing the extract solution prepared in Example 1 was added at a concentration of 0.5 wt% (as a treatment group) and cultured for 24 hours and 48 hours respectively. The wells were washed with phosphate buffered saline (PBS) for three times, 100 μl of antibody-POD conjugate solution was added to the wells and left for 1 hour, then the wells were washed three times with PBS, 100 μl of substrate solution (TMB) was added to the wells and left at 25°C for 30 minutes. 100 μl of 1NH 2 SO 4 Add to the wells, and use an ELISA plate reader to measure the expression level of collagen at an absorbance of 450 nm.

[0165] At this time, the collagen production expression level of the treatment group was calculated based on the relative absorbance difference between the treatment group and the DMSO-treated control group, and the results were obtained. Figure 2 .

[0166] according to Figure 2 It can be seen that compared with the control group, the collagen expression in the treatment group increased significantly regardless of whether it was cultured for 24 hours or 48 hours.

[0167] That is, the extract provided in the present application has the effect of promoting the increase of collagen expression.

[0168] 3. Inhibitory effect on MMP-1 expression in dermal fibroblasts

[0169] After dermal fibroblasts were seeded into 6-well plates, they were cultured for 24 hours to allow the cells to adhere to the plate wall. The culture medium was removed and washed with phosphate buffered saline (PBS). Culture was performed for 24 hours in a culture medium containing the extract solution prepared in Example 1 with an added concentration of 0 (as a control group), 0.1wt% (as a treatment group), and 0.5wt% (as a treatment group). After removing the culture medium, the cells were washed with PBS. RNA was extracted using the Takara MiniBEST Universal RNA Extraction Kit. 1ug of total RNA was used for each sample to synthesize cDNA.

[0170] Real-time polymerase chain reaction was performed using the synthesized cDNA. Specifically, SYBR Green fluorescent dye was used for quantification of the real-time polymerase chain reaction. The specific primer sequences of human MMP-1 are forward primer 5'-GGG GAG ATCGGG AGG ACA AC-3' and reverse primer 5'-GGG TAC ATC AAA GCC CCG AT-3'. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal reference control group for the PCR reaction. The specific polymerase chain reaction (PCR) conditions were 95°C for 15 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 45 seconds. The results are shown in Figure 3 .

[0171] Reference Figure 3 It can be confirmed that the extract provided in Example 1 can inhibit the expression of the MMP-1 gene that causes wrinkles. Therefore, it can be confirmed that the extract in Example 1 can inhibit the formation of skin wrinkles.

[0172] 4. Promoting effect of TIMP-1 expression in dermal fibroblasts

[0173] After dermal fibroblasts were seeded into 6-well plates, they were cultured for 24 hours to allow the cells to adhere to the plates. The culture medium was removed and washed with phosphate buffered saline (PBS). The cells were cultured for 24 hours in a culture medium containing the extract solution prepared in Example 1 with the added concentrations of 0 (as a control group), 0.1wt% (as a treatment group), and 0.5wt% (as a treatment group). After removing the culture medium, the cells were washed with PBS. RNA was extracted using the Takara MiniBESTuniversal RNA extraction kit. 1ug of total RNA was used for each sample to synthesize cDNA. Real-time polymerase chain reaction (Real time PCR) was performed using the synthesized cDNA. Specifically, SYBR Green fluorescent dye was used for quantitative real-time polymerase chain reaction. The specific primer sequences for human TIMP-1 are forward primer 5'-ACT TCC ACA GGTCCC ACA AC-3' and reverse primer 5'-AAA CAG GGA AAC ACT GTG CAT-3'. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal reference control group for the PCR reaction. The specific polymerase chain reaction (PCR) conditions were 95°C for 15 minutes, followed by 95°C for 15 minutes and 60°C for 45 seconds for a total of 40 cycles. The results are shown in Figure 4 .

[0174] Reference Figure 4 It can be confirmed that the extract provided in Example 1 can promote the expression of TIMP-1 gene, and promoting the expression of gene TIMP-1 can increase the production of collagen.

[0175] In summary, the extract provided in Example 1 can prevent and improve wrinkles.

[0176] Test Example 3: H 2 O 2 Antioxidant effect of oxidative stress

[0177] 1. Using H in the HaCaT cell line of human keratinocytes 2 O 2 The active oxygen inhibition effect was evaluated to evaluate the antioxidant effect.

[0178] HaCaT cells were cultured in a 96-well plate at 3 × 10 cells per well using DMEM medium containing 10% bovine serum. 4 100 μl of cells / well were inoculated and cultured for 24 hours. After that, each well was replaced with serum-free medium containing the samples shown in Table 7. After 24 hours of culture, the cells were washed with HBSS. 2The cultured cells were treated with DCFDA (2',7'-dichlorodihydrofluorescein diacetate) for 30 minutes, and then the overstained fluorescence was washed with HBSS (Hanks' Balanced Salt Solution). 2 O 2 The treated group and the untreated group 2 O 2 After 10 minutes of treatment, the cells were washed with HBSS and the fluorescence value was measured at 492 nm using a microplate reader (spark 10M Elisa reader). After removing the buffer, 50 μl of 0.5% Triton X-100 and 50 μl of BCA solution were added, and the protein expression level was measured using the BCA protein quantification method. ROS activity was calculated using the fluorescence value and protein amount measured previously. The results are shown in Table 7 and Figure 5 . Among them, 100 μM H 2 O 2 The ROS generation amount of the treated group was set as 100%, and the percentage yield of ROS generation amount of each sample corresponding to the treated group was calculated as the ROS generation amount.

[0179] Table 7 Protein expression level

[0180]

[0181]

[0182] Figure 5 Medium(-)H 2 O 2 Indicates that no H 2 O 2 Example 1 treated with (+) 100 μM H 2 O 2 Indicates that 100 μM H 2 O 2 For the treated Example 1, the abscissa represents the concentration of the added extract solution of Example 1.

[0183] As shown in Table 7 and Figure 5 As shown in the results, it can be seen that the extract of the present application has excellent inhibitory factor H 2 O 2 The ability to inhibit the active oxygen produced by oxidative stress. According to Comparative Examples 1 and 7, it can be seen that the extract composed of the above raw materials in this application has the ability to inhibit the active oxygen produced by H 2 O 2Furthermore, the ability to inhibit active oxygen generated by oxidative stress is significantly higher in Examples 1-7 of the present application than in Comparative Examples 1-7, indicating that the raw materials in a specific ratio used in the present application have a good synergistic effect with each other and significantly improve their antioxidant capacity.

[0184] 2. Confirm the cause of H 2 O 2 Restoration effect of reduced NQO1 gene expression caused by oxidative stress

[0185] In order to evaluate the detoxification effect on oxidative stress, NQO1 (NAD(P)H:quinoneoxidoreductase 1) gene expression experiment was performed.

[0186] Human keratinocytes (HaCaT) were added to DMEM medium containing 10% (v / v) bovine serum and plated on a 6-well plate at 1.5 × 10 6 2 ml of cells / well were inoculated and cultured for 24 hours. After that, each well was replaced with serum-free medium and treated with samples as shown in Table 8. After 24 hours of culture, 10 μM H 2 O 2 After washing with PBS, ( USA) to treat the cultured cells for cell lysis and then use the manufacturer's RNA was isolated using the protocol provided by After the isolated RNA was quantified by a fluorimeter, cDNA was synthesized and real-time polymerase chain reaction (Real-time PCR) was performed. cDNA synthesis was performed using a cDNA synthesis kit (qPCRBIO cDNA Synthesis Kit, PCRBIOSYSTEMS, London, UK), and the experiment was performed according to the method of the kit. Real-time polymerase chain reaction was performed by amplifying the gene using a real-time polymerase chain reaction kit (2x qPCRBIO SyGreen Blue mix Lo-ROX, PCRBIOSYSTEMS, London, UK), and the amplified product was quantitatively analyzed. The NQO1 and β-actin primers used for PCR were synthesized by Cosmo Gene Tech (Korea) and used.

[0187] The results are shown in Table 8. Figure 6 .

[0188] Table 8 Test results

[0189]

[0190]

[0191] As shown in Table 8 and Figure 6 As shown in the results, the restoration effect of NQO1 gene expression in Examples 1-7 of the present application is significantly higher than that in Comparative Examples 1-7, indicating that the raw materials in a specific ratio used in the present application have a good synergistic effect with each other and can significantly improve the restoration effect of anti-NQO1 gene expression.

[0192] In summary, the extract of the present application has antioxidant function.

[0193] Test Example 4

[0194] 1. Confirm the effect of reducing NO production and cell survival rate in inflammatory response induced by lipopolysaccharide (LPS)

[0195] The inhibitory effect of LPS on the production of NO in mouse-derived macrophage Raw264.7 cells was evaluated. Specifically, the Raw264.7 cells, which are mouse macrophages, were treated with lipopolysaccharide (LPS) (Sigma, St. Louis, MO) to measure the ability to inhibit the activity of inducible Nitric Oxide Synthase (iNOS) to confirm the inhibitory effect on artificially induced inflammation.

[0196] 8×10 5 Raw264.7 cells were suspended in 10% (v / v) FBS-DMEM (WELLGIN Co., Ltd.) medium, inoculated and attached to a 24-well cell culture plate. One day later, the medium was replaced with one containing 0.1 μg / ml lipopolysaccharide. After inducing inflammation, the samples shown in Table 9 were treated and cultured for 24 hours, and the supernatant was recovered. 100 μl was added to each 96-well plate, and the same Griess' reagent (Sigma, St. Louis, MO) was added. After gently shaking at room temperature for 10 minutes, the absorbance was measured at 570 nm. The NO generation amount of the sample of the group treated with lipopolysaccharide alone was set to 100%, and the NO percentage yield of each sample corresponding to the treatment group was calculated as NO activity.

[0197] In addition, after removing the sample treatment medium, the cells were treated with a 0.25 mg / ml MTT solution. After 4 hours, the MTT was removed and the cells were treated with DMSO. The absorbance was measured at 570 nm, and the cell survival rate was calculated according to the following mathematical formula to evaluate the toxicity of each sample to the cells.

[0198] The following table 9 and Figure 7 Results shown.

[0199] Wherein, cell survival rate (%) = (sample absorbance value / control group absorbance average) × 100.

[0200] Table 9 Test results

[0201]

[0202]

[0203] As shown in Table 9 and Figure 7 As shown in the results, it is proved that the extract of the present application has no toxicity to the macrophage Raw264.7.

[0204] And according to Comparative Examples 1 to 7 in Table 9, Comparative Examples 1 and 7 have better NO generation inhibition effect than other comparative examples because they contain all raw materials. It can be seen that the extracts composed of the above raw materials in the present application cooperate with each other and have better NO generation inhibition ability. And further, for the ability to inhibit NO generation, Examples 1-7 of the present application are significantly higher than Comparative Examples 1-7, indicating that the raw materials used in the present application have good synergistic effects with each other, which can significantly improve its NO generation inhibition ability and significantly improve the anti-inflammatory effect.

[0205] 2. Confirmation of the ability to suppress iNOS gene expression in LPS-induced inflammatory response

[0206] Raw264.7 cells, which are mouse macrophages, were treated with lipopolysaccharide to artificially increase the gene expression of iNOS, an inflammatory-related enzyme, and then the expression inhibition of the mixed extract sample according to the present application was measured by the real-time PCR method.

[0207] 2×10 6 The cells were suspended in 10% FBS-DMEM medium, seeded and attached to a 60 mm culture dish. One day later, the medium was replaced with 0.1 μg / ml lipopolysaccharide, and after inducing inflammation, the samples were treated as shown in Table 10 and cultured for 24 hours. The cell medium was removed and the cells were treated with ( USA) for cell lysis and then the manufacturer RNA was isolated using the protocol provided by After the isolated RNA was quantified by a fluorimeter, cDNA was synthesized and real-time polymerase chain reaction (Real-time PCR) was performed. The cDNA synthesis was performed using a cDNA synthesis kit (qPCRBIO cDNA Synthesis Kit, PCRBIOSYSTEMS, London, UK), and the experiment was performed according to the method of the kit. Real-time polymerase chain reaction was performed by quantitatively analyzing the amplified product after amplifying the gene using a real-time polymerase chain reaction kit (2x qPCRBIO SyGreen Blue mix Lo-ROX, PCRBIOSYSTEMS, London, UK). The iNOS and glyceraldehyde phosphate dehydrogenase primers (GAPDH primer) used for PCR were synthesized and used by Cosmo Gene Technology Co., Ltd. (South Korea). Among them, the iNOS mRNA expression level of the sample of the group treated with lipopolysaccharide alone was set to 100%, and the percentage yield of the iNOS mRNA expression level of each sample corresponding to the treatment group was calculated as the iNOS mRNA expression level.

[0208] The results are shown in Table 10 and Figure 8 shown.

[0209] Table 10 Results of the iNOS gene expression inhibition test

[0210]

[0211] As shown in Table 10 and Figure 8 As shown in the results, since the extract of the present application has an excellent iNOS gene expression inhibitory effect, it can be confirmed that it has anti-inflammatory function.

[0212] And according to Comparative Examples 1 to 7 in Table 10, Comparative Examples 1 and 7 have better iNOS gene expression inhibition effects than other comparative examples due to the presence of all raw materials. And further, in terms of the ability to inhibit NO generation, Examples 1-7 of the present application are significantly higher than Comparative Examples 1-7, indicating that the raw materials used in the present application have good synergistic effects with each other, which can significantly improve its ability to inhibit iNOS gene expression and significantly improve the anti-inflammatory effect.

[0213] 3. Confirmation of the inhibitory effect of COX-2 gene expression in LPS-induced inflammatory response

[0214] Raw264.7 cells, which are mouse macrophages, were treated with lipopolysaccharide to artificially increase the gene expression of COX-2, an inflammatory enzyme, and then the expression inhibition of the mixed extract sample according to the present application was measured by the real-time PCR method.

[0215] 2×10 6 Raw264.7 cells were suspended in 10% FBS-DMEM medium, seeded and attached to a 60 mm culture dish. One day later, the medium was replaced with 0.1 μg / ml lipopolysaccharide, and after inducing inflammation, the samples were treated as shown in Table 11 and cultured for 24 hours. The cell medium was removed and the cells were washed with ( USA) for cell lysis and then the manufacturer RNA was isolated using the protocol provided by After the isolated RNA was quantified by a fluorimeter, cDNA was synthesized and real-time polymerase chain reaction (Real-time PCR) was performed. cDNA synthesis was performed using a cDNA synthesis kit (qPCRBIO cDNA Synthesis Kit, PCRBIOSYSTEMS, London, UK), and the experiment was performed according to the method of the kit. Real-time polymerase chain reaction was performed by quantitatively analyzing the amplified product after amplifying the gene using a real-time polymerase chain reaction kit (2x qPCRBIO SyGreen Blue mixLo-ROX, PCRBIOSYSTEMS, London, UK). The COX-2 and glyceraldehyde phosphate dehydrogenase primers (GAPDH primer) used for PCR were synthesized and used by Cosmo Gene Technology Co., Ltd. (South Korea). Among them, the COX-2 mRNA expression level of the sample of the group treated with lipopolysaccharide alone was set to 100%, and the percentage yield of the COX-2 mRNA expression level of each sample corresponding to the treatment group was calculated as the COX-2 mRNA expression rate.

[0216] The results are shown in Table 11 and Fig. 9 shown.

[0217] Table 11 Results of COX-2 gene expression inhibition experiment

[0218]

[0219] As shown in Table 11 and Fig. 9 As shown in the results, the extract of the present application has an excellent COX-2 gene expression inhibitory effect, and it can be confirmed that it has anti-inflammatory function.

[0220] And according to Comparative Examples 1 to 7 in Table 11, it can be seen that the extracts composed of the above raw materials of the present application cooperate with each other and have a better COX-2 gene expression inhibition effect. And further, for the ability to inhibit NO generation, Examples 1-7 of the present application are significantly higher than Comparative Examples 1-7, indicating that the raw materials used in the present application have a good synergistic effect with each other, which can significantly improve its ability to inhibit COX-2 gene expression and significantly improve the anti-inflammatory effect.

[0221] In summary, Experimental Example 4 determined that the extract provided by the present application has a better anti-inflammatory effect.

[0222] In summary, the Chinese medicine composition and extract provided by the present application all utilize the specific raw materials of Forsythia suspensa, Tribulus terrestris, Acanthopanax senticosus, Polygonatum odoratum, Citrus aurantium and Ginseng to cooperate with each other and enhance synergy, and have the ability to inhibit MMP-1 gene expression, promote TIMP-1 expression, inhibit reactive oxygen generation, promote NQO1 gene expression reduced by oxidative stress, inhibit NO generation and inhibit iNOS expression. It not only has antioxidant, anti-wrinkle and anti-inflammatory effects, but is also non-toxic, and therefore can be used in related fields such as medicines, cosmetics and health products.

[0223] The above are only specific embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A Chinese medicine composition for anti-inflammatory, anti-oxidative and / or anti-wrinkle effects, characterized in that: The invention comprises the following ingredients: 100 parts by weight of forsythia fruit, 100-150 parts by weight of tribulus terrestris fruit, 75-125 parts by weight of acanthopanax senticosus, 100-150 parts by weight of polygonatum odoratum, 25-50 parts by weight of immature aurantium and 50-75 parts by weight of ginseng.

2. An extract, characterized in that The extractant is obtained by extracting the traditional Chinese medicine composition according to claim 1 with an extractant, wherein the extractant is an ethanol aqueous solution, and the volume fraction of ethanol in the ethanol aqueous solution is 25-35%.

3. A method for preparing an extract, characterized in that: Obtained by extracting the Chinese medicine composition according to claim 1 with an extractant; Wherein, the extractant is an ethanol aqueous solution, and the volume fraction of ethanol in the ethanol aqueous solution is 25-35%.

4. The preparation method according to claim 3, characterized in that: The extraction comprises: extracting by ultrasonic wave for 1.5-3h.

5. Use of the Chinese medicine composition according to claim 1, the extract according to claim 2, or the extract prepared by the preparation method according to any one of claims 3 to 4 in the preparation of anti-inflammatory, antioxidant and / or anti-wrinkle preparations, characterized in that: The preparation includes any one of cosmetics and medicines.

6. A cosmetic, characterized in that: It includes the Chinese medicine composition as claimed in claim 1, the extract as claimed in claim 2, or the extract prepared by the preparation method as claimed in any one of claims 3-4.

7. The cosmetic according to claim 6, characterized in that: The extract is added in an amount of 0.01 to 5 wt % in the cosmetic.

8. The cosmetic according to claim 6, characterized in that: The extract is added in an amount of 0.01 to 3 wt % in the cosmetic.

9. A medicine, characterized in that: It includes the Chinese medicine composition as claimed in claim 1, the extract as claimed in claim 2, or the extract prepared by the preparation method as claimed in any one of claims 3-4.

10. The drug according to claim 9, characterized in that The amount of the extract added to the medicine is 0.001-30wt%.

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