KR102142060B1 - Anti-inflammatory agent containing caesalpinia sappan microwave extract - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition comprising a bovine microwave extract as an active ingredient. The bovine extract has no cell microwave toxicity and uses 70% ethanol aqueous solution, which is recognized as safe for existing edible or medicinal plants, as an extraction solvent, so that side effects and resistance are not a problem, as well as mouse-derived macrophages RAW264. As a result of experiments using 7 cells, it was confirmed that the inhibitory effect of the production and expression of inflammatory mediators by LPS was remarkably superior to that of the conventional solvent extraction method. It can be applied as an anti-inflammatory and anti-inflammatory cosmetic composition that suppresses inflammation.

Description

ANTI-INFLAMMATORY AGENT CONTAINING CAESALPINIA SAPPAN MICROWAVE EXTRACT WITH ANTI-INFLAMMATORY AGENT CONTAINING

The present invention relates to an anti-inflammatory composition comprising a plant extract having an anti-inflammatory effect, specifically, bovine ultra-high extract as an active ingredient.

Inflammation is an immune response that occurs locally to minimize damage and restore the damaged area to its original state when cells or tissues are damaged or destroyed by various factors, such as harmful substances or organisms that come in from outside, protecting the living body And is a useful defense mechanism to remove products produced by tissue damage. As a result of the defense mechanism, pain, swelling, redness or fever may occur as a result, resulting in dysfunction. The factors causing inflammation include trauma, burns, frostbite, radioactive factors, physical factors, chemical factors such as acids, and immunological factors caused by antibody reactions. It may be caused by.

In the normal case, the inflammation acts to neutralize or eliminate the onset factors through an inflammatory reaction in vivo and restore the normal structure and function by regenerating the upper tissue, but the degree of inflammation becomes a certain level or more or becomes chronic and chronic inflammation It becomes a problem when it progresses to a disease state such as. In addition to being able to observe the inflammatory response in almost any disease among clinical diseases, it is known that enzymes related to the inflammatory response play an important role in carcinogenesis.

According to the recent findings, it is known that the progress of the inflammatory reaction in the body is related to the activity of the COX (cyclooxygenase) enzyme. The COX enzyme is a major enzyme involved in the biosynthesis of prostaglandin present in vivo (Smith et al., J. Biol. Chem., 271, 33157 (1996)), two types of isomers, COX-1 and COX It is known that -2 exists. The COX-1 is constantly present in tissues such as the stomach and kidneys, and is involved in maintaining normal homeostasis, while the COX-2 is involved in mitogens or cytokines during inflammation or other immune responses. It is an enzyme that is transiently and rapidly expressed in cells.

Another powerful inflammatory mediator, nitric oxide (NO), is produced from L-arginine by NO synthetase (NOS), and has many types due to external stress such as UV or substances such as endotoxin or cytokine. It is produced in cells. The inflammatory stimuli as described above may increase the expression of inducible NOS (iNOS) in the cell, and thereby induce NO production in the cell, thereby activating macrophages to cause an inflammatory reaction.

Therefore, in recent years, studies on substances capable of inhibiting the production of NO have been conducted for effective inflammation relief. However, in the case of the anti-inflammatory substances developed by these studies, some side effects are problematic. For example, nonsteroidal anti-inflammatory drugs used in the treatment of chronic inflammatory diseases such as acute or rheumatoid arthritis are known to exhibit side effects such as gastrointestinal disorders by inhibiting the COX-2 enzyme as well as the COX-1 enzyme. .

On the other hand, cosmetics used in everyday life are products used to protect the skin and clean the skin, but when looking at the cosmetic composition, ingredients different from the purpose of skin protection are used as essential for product formation. Examples include surfactants, preservatives, fragrances, sunscreens, pigments, and other ingredients used to impart efficacy or effectiveness. It is known that ingredients used in the manufacture of cosmetics generally cause various side effects such as inflammation, rash or edema on the skin.

In addition, if sebum components, sweat components and fatty acids in the cosmetic components emitted from the body, higher alcohols, proteins, etc., are decomposed into highly toxic substances by skin-existing bacteria present on the skin, skin inflammation may be caused by them. It is well known that skin irritation is caused by ultraviolet rays from the sun.

As such, the cause of skin side effects in cosmetics is always latent, and various studies have been conducted to solve them. Materials that have been used for the purpose of alleviating irritation and inflammation such as erythema and edema to date include non-steroidal systems such as flufenamic acid, ibuprofen, benzydamine, and indomethacin. , Steroids include prednisolone, dexamethasone, allantoin, azrene, ε-aminokipronic acid, hydrocortisone, licorice acid and its derivatives (β-glycylic acid, glycylic acid derivatives) It is known to have anti-inflammatory effects.

However, indomethacin, which is generally used as an anti-inflammatory agent, is a substance that cannot be used in cosmetics, and the amount of hydrocortisone is limited, and licorice acid and its derivatives are difficult to stabilize or have poor solubility. Due to the fact that it is difficult to obtain a practical effect, the anti-inflammatory agents known so far have mostly problems in terms of skin safety and stability when blending cosmetics, and thus their use is limited.

Therefore, it is a natural substance-derived material that has no or little risk of side effects or cytotoxicity, and can effectively suppress the production of NO, so it has an excellent anti-inflammatory effect, and can suppress inflammation without side effects as well as without limiting its content. There is a need to develop substances that can be used in cosmetics.

In particular, in recent years, research and development on cosmetics using natural raw materials has been actively conducted in order to meet the needs of consumers.

KR 10-1434409 B KR 2016-0147540 A KR 2015-0019380 A

In order to meet the above-mentioned needs, the present invention aims to provide an anti-inflammatory composition comprising as an active ingredient a plant extract that is less likely to cause problems associated with side effects.

In addition, unlike conventional anti-inflammatory substances, side effects, such as natural plants that can be used for edible or medicinal use are not a problem, as well as excellent safety, can effectively suppress inflammatory cytokines related to inflammation, and suppress NO production It is an object to provide an anti-inflammatory or cosmetic composition comprising a plant extract as an active ingredient.

In order to achieve the above object, the present invention is caesalpinia sappan ) It provides an anti-inflammatory composition comprising a microwave extract as an active ingredient.

The small-wave microwave extract may be obtained by adding an extraction solvent to at least one selected from the group consisting of water, alcohols having 1 to 5 carbons and mixtures thereof, and extracting them by ultra-short microwave extraction.

Specifically, the small-wave microwave extract may be extracted by adding an aqueous solution of ethanol to the stem of the pine tree and extracting it under a microwave extraction method under conditions of 2,000 Hz to 3,000 Hz.

More specifically, the ultra-high extract of the bovine can be extracted by adding a 70% ethanol aqueous solution to the bovine trunk and extracting it at 2,450 Hz under microwave extraction.

The bovine ultra-high frequency extract can effectively suppress NO secretion, control various cytokines related to inflammation, and are less likely to cause side effects because they are derived from natural products, compared to the existing bovine solvent extract. It was confirmed.

The anti-inflammatory composition of the present invention may be provided as an active ingredient of an anti-inflammatory pharmaceutical composition.

In this aspect, the present invention is Caesalpinia sappan ) It provides a pharmaceutical composition for anti-inflammatory, including a microwave extract as an active ingredient.

The small-wave microwave extract may be obtained by adding an extraction solvent to at least one selected from the group consisting of water, alcohols having 1 to 5 carbons and mixtures thereof, and extracting them by ultra-short microwave extraction.

Preferably, the small-wave microwave extract may be extracted by adding an ethanol aqueous solution to the stem of the pine tree and extracting it by microwave extraction under conditions of 2,000 Hz to 3,000 Hz.

More preferably, the small-wave microwave extract may be extracted by adding a 70% ethanol aqueous solution to the small-tree stem microwave extract at 2,450 Hz under the condition of 2,450 Hz.

In addition, the anti-inflammatory composition of the present invention may be provided as an active ingredient of the cosmetic composition for anti-inflammatory purposes or an anti-inflammatory cosmetic composition, that is, an active ingredient of an anti-inflammatory cosmetic composition.

In this aspect, the present invention is Caesalpinia sappan ) It provides a cosmetic composition having an anti-inflammatory effect that includes microwave extract as an active ingredient.

The small-wave microwave extract may be obtained by adding an extraction solvent to at least one selected from the group consisting of water, alcohols having 1 to 5 carbons and mixtures thereof, and extracting them by ultra-short microwave extraction.

Preferably, the small-wave microwave extract may be extracted by adding an ethanol aqueous solution to the stem of the pine tree and extracting it by microwave extraction under conditions of 2,000 Hz to 3,000 Hz.

More preferably, the small-wave microwave extract may be extracted by adding a 70% ethanol aqueous solution to the small-tree stem microwave extract at 2,450 Hz under the condition of 2,450 Hz.

In another aspect, the present invention is a joiner preparation step of preparing the joiner stem pulverized by washing and drying the joiner stem; And adding a 70% ethanol aqueous solution to the lumber stem pulverized product and performing ultra-high frequency extraction under 2,450 Hz conditions.

The present inventors, while studying the components derived from natural products having an anti-inflammatory effect, confirmed that the pine extract has an anti-inflammatory effect, and through further research, the pine ultra-high extract produced by the extraction method using microwave was used in the conventional heating extraction method or solvent extraction method. Through the result that it is possible to significantly suppress the cytokines associated with inflammation causing inflammation and to effectively suppress the secretion of inflammatory mediators such as NO, which can directly induce inflammation, as compared to the manufactured pine tree solvent extract. , Completed the present invention by confirming that the anti-inflammatory effect of cedar ultrafine is significantly superior to other extraction methods.

Hereinafter, the present invention will be described in more detail.

The present invention relates to an anti-inflammatory composition comprising a bovine extract, preferably a bovine ultra-high extract as an active ingredient.

The joiner ( Caesalpinia sappan ) is an evergreen tree belonging to the legume family, and is also called firewood, red-eye, or red-eye.

The small tree is a plant mainly grown in the tropics, and grows to a height of about 5 to 9 m, has small thorns in the water, and small thorns are grayish green and have holes. The leaves are two times idols, and the mail faces each other and has a long oval shape. Flowers bloom in May-June, yellow flowers, and fruit in September-October.

The red-yellow wood part of the joiner was used as a red dye, and the root was used as a yellow dye, and was mainly used as a dye for dyeing red in the Joseon Dynasty. Since it was not calculated in Korea, more than 70,000 roots were imported during the nine years of King Sejong.

The joiner has been reported to have anti-oxidant effect, antibacterial effect, anti-inflammatory effect, or vasodilator effect, and has been used as a medicine since it has efficacy such as analgesic, edema, and haemorrhagic effects in oriental medicine. Specifically, in the oriental medicine, there is an efficacy of hemostasis, hemostasis, oral blood, analgesic, and edema, and heart material is used as a medicine, and mainly used for symptoms such as gynecological cardiac abdominal pain, menopause, postpartum blood abdominal pain, tetanus or carbuncle. come.

The bovine extract may be prepared by performing an extraction method on a leaf, a branch, a stem, a flower, a fruit, a root or a mixture thereof, or a pulverized product of the bovine, as in a method for preparing a normal plant extract, and is preferable. For example, it may be manufactured by performing an extraction method on a stem or wood of a joiner.

The joiner extract may be prepared according to a method of extracting using microwave or microwave in terms of the remarkable effect of the conventional plant extract manufacturing method, for example, leaf, branch, stem, flower, fruit, root or These mixtures or pulverized products thereof, preferably, may be prepared by adding an extractant to the pulverized product of the stem and extracting it by microwave extraction, or by fractionating by adding a fractional solvent to the microwave extract prepared by extraction with an extraction solvent.

The microwave refers to an electromagnetic wave having a very high frequency, and is also called a microwave. When classified by frequency, electromagnetic waves are long waves having a frequency of 30 to 300 kHz, medium waves having a frequency of 300 to 3,000 kHz, centimeter waves having a frequency of 3,000 MHz (3 GHz) to 30 GHz, and millimeter waves having a frequency of 30 to 300 GHz. It can be classified as a submillimeter wave of 300 GHz or more. Of these, the electromagnetic waves having a frequency of 300 MHz to 30 GHz are called microwaves or microwaves.

In addition, in terms of wavelength, radio waves with a wavelength range of 1 mm to 1 m are called microwaves or microwaves.

Since the microwave has a short wavelength, it has almost similar properties to light or light waves, and thus has straight, reflective and absorbing properties. In addition, it has a strong sterilizing power, and is well absorbed by plants or water to generate heat. Therefore, in addition to being widely used for communication (transmission of radio waves), it is used for heating food by ultra-short dielectric heating, “defrosting frozen food,” simple sterilization of food, and “sterilization” of pests in food. It was applied to microwave ovens by applying these characteristics.

The microwave extraction method refers to an extraction method in which an extraction solvent is added to an object to be extracted using the microwave, and the microwave is processed to extract it.

The extraction solvent may be one or more selected from the group consisting of water and organic solvents. The organic solvent may be an alcohol having 1 to 5 carbon atoms, dilution of the alcohol, a polar solvent such as ethyl acetate or acetone, and a non-polar solvent of ether, chloroform, benzene, hexane or dichloromethane, or a mixed solvent thereof. The alcohol having 1 to 5 carbon atoms may be methanol, ethanol, propanol, butanol, isopropanol, and the like, but is not limited thereto. In addition, the alcohol dilution water may be a 50% (v/v) to 99.9% (v/v) of alcohol diluted in water.

The extracting solvent of the pine tree extract of the present invention may preferably be water, alcohol having 1 to 5 carbons or diluted alcohol, more preferably water, alcohol having 1 to 4 carbons or diluted alcohol, and even more preferably It may be an aqueous ethanol solution or ethanol. The ethanol aqueous solution is preferably 50% (v/v) to 99.9% (v/v) ethanol aqueous solution, more preferably 60% (v/v) to 80% (v/v) ethanol aqueous solution, even more preferably May be 70% (v/v) ethanol aqueous solution.

The extraction process is, for example, in the frequency conditions of 1,500 Hz to 3,500 Hz, 2,000 Hz to 3,000 Hz, or 2,400 Hz to 2,500 Hz and output conditions of 500 W to 1,000 W or 600 W to 800 W or 650 W to 750 W Can be performed. In addition, the extraction time is not particularly limited, but may be 5 minutes to 60 minutes, or 7 minutes to 45 minutes, or 10 minutes to 20 minutes.

In addition, the extract extracted by the ultra-short method can be further subjected to the fractionation process with any one solvent selected from the group consisting of butanol, hexane, methylene chloride, acetone, ethyl acetate, ethyl ether, chloroform, water, and mixtures thereof. have. At the time of fractionation, two or more solvents may be used, and each solvent extract may be prepared by sequentially using or mixing depending on the polarity of the solvent.

The prepared extract or the fraction obtained by performing the fractionation process may then be filtered or concentrated or dried to remove the solvent, and all of filtration, concentration and drying may be performed. Specifically, the filtration may use a filter paper or a reduced pressure filter, the concentration may be concentrated under reduced pressure using a reduced pressure concentrator, for example, a rotary evaporator, and the drying may be performed, for example, by freeze drying.

According to an embodiment of the present invention, it was confirmed that the bovine ultra-high frequency extract can effectively suppress cytokines related to inflammation causing inflammation, and can effectively suppress the secretion of NO, which can directly induce inflammation. .

Therefore, the anti-inflammatory composition comprising the bovine ultra-high extract as an active ingredient may be used to suppress inflammation or to treat, improve or prevent inflammation.

The inflammation is a concept including a general inflammatory disease, for example, the inflammatory disease may be one or more selected from the group consisting of various dermatitis, allergies, systemic lupus erythematosus, retinitis, gastritis, hepatitis, enteritis, pancreatitis, arthritis and nephritis. The allergies include anaphylaxis, allergic rhinitis, asthma, allergic conjunctivitis, allergic dermatitis, atopic dermatitis, contact dermatitis, urticaria, and insect allergy. , Food allergies and drug allergies.

Therefore, the anti-inflammatory composition of the present invention may be applied as a composition for the treatment or prevention of inflammatory diseases, or as a food composition for preventing or improving the inflammatory diseases. The food composition may be, for example, a health functional food composition for preventing or improving the inflammatory disease.

The health functional food uses food, physical, biochemical, and biotechnological techniques, etc. to provide added value to act and express the function of the food for a specific purpose, or control the biological defense rhythm of the food group or food composition, prevent disease and recover Refers to a food product that has been designed and processed to sufficiently express the body control function. The dietary supplement may include a food-acceptable food supplement additive, and may further include suitable carriers, excipients, and diluents commonly used in the manufacture of dietary supplements.

The health functional food composition for preventing or improving the inflammatory disease of the present invention comprises 0.001% by weight to 99.9% by weight or 0.01% by weight to 50% by weight or 0.1% by weight to 30% by weight or 0.1% by weight of the whole bovine microwave extract. % To 15% by weight.

The anti-inflammatory composition comprising the bovine ultra-high extract as an active ingredient can be applied directly to animals, including humans. The animal is a group of organisms corresponding to the plant, which mainly consumes organic matter as nutrients, and refers to a differentiation of the digestive organs, excretory organs, and respiratory organs, and preferably may be mammals, more preferably humans.

The cedar microwave extract may be used alone in the anti-inflammatory composition, and may further include other pharmacologically acceptable carriers, excipients, diluents or sub-components. More specifically, when the composition containing the cedar microwave extract is used as a medicament, or for pharmaceutical or pharmaceutical use, the cedar microwave extract is mixed with a pharmaceutically acceptable carrier or excipient according to a conventional method, or It can be used diluted with a diluent.

In this case, the content of the ultra-high frequency extract in the composition may be 0.001% by weight to 99.9% by weight, 0.1% by weight to 99% by weight, or 1% by weight to 50% by weight, but is not limited thereto. Depending on the method, the content of the compound may be appropriately adjusted to a desired content and used.

Examples of the pharmaceutically acceptable carrier, excipient, or diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, propylhydroxybenzoate, talc, magnesium stearate and mineral oil , Dextrin, calcium carbonate, propylene glycol, liquid paraffin, and one or more selected from the group consisting of physiological saline, but is not limited thereto, and a common carrier, excipient, or diluent may be used. In addition, the pharmaceutical composition is a conventional filler, extender, binder, disintegrant, anti-coagulant, lubricant, wetting agent, pH adjusting agent, nutrient, vitamin, electrolyte, alginic acid and salts thereof, pectic acid and salts thereof, protective colloids, glycerin , Fragrances, emulsifiers or preservatives. The components may be added independently or in combination to the active ingredient, bovine pine extract.

In addition, the composition of the present invention may further include a substance that is recognized as having a known anti-inflammatory effect in addition to the active ingredient, for example, a substance used as a COX-2 inhibitor, NO inhibitor, or anti-inflammatory agent.

When the composition is used as a pharmaceutical, the administration method may be oral or parenteral, and for example, it may be administered through various routes including oral, transdermal, subcutaneous, intravenous, or intramuscular.

In addition, the formulation of the composition may vary depending on the method of use, and formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. Can be. In general, solid preparations for oral administration include tablets, pills, soft or hard capsules (CAPSULES), pills (PILLS), powders (POWDERS) and granules (GRANULES), etc. Excipients, for example, may be prepared by mixing starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients. Liquid preparations for oral use include suspensions, solvents, emulsions and syrups, and water, liquid paraffin, and other common excipients, such as wetting agents, wetting agents, Sweeteners, fragrances, preservatives, and the like may be included.

Forms for parenteral administration include cream, lotions, ointments, warnings, liquids, aerosols, aerosols, fluid extracts, elixirs (ELIXIR), acupuncture (INFUSIONS), sachet (SACHET), patch (PATCH) or injection (INJECTIONS).

Furthermore, the compositions of the invention may be formulated using suitable methods known in the art or using methods disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA. Can.

The dosage of the composition may be determined in consideration of the administration method, the age, sex of the patient, the severity of the patient, the condition, the absorption of the active ingredient in the body, the inactive rate and the drugs used in combination, for example, based on the daily active ingredient 0.1 mg/kg (weight) to 500 mg/kg (weight), 0.1 mg/kg (weight) to 400 mg/kg (weight) or 1 mg/kg (weight) to 300 mg/kg (weight) It can be administered as, and can be administered once or several times. The above dosage does not limit the scope of the invention in any aspect.

The present invention also provides an anti-inflammatory composition comprising the bovine microwave extract as an active ingredient or an anti-inflammatory agent comprising the bovine microwave extract as an active ingredient or an anti-inflammatory pharmaceutical composition comprising the bovine microwave extract as an active ingredient.

The joiner extract may be preferably a joiner microwave extract, more preferably a joiner ethanol 70% aqueous solution microwave extract.

The anti-inflammatory pharmaceutical composition may include the active ingredient alone, and may further include a pharmaceutically acceptable carrier or excipient depending on the formulation, method of use, and purpose of use. When provided as a mixture, the active ingredient may be included in an amount of 0.1% to 99.9% by weight relative to the entire anti-inflammatory pharmaceutical composition, but is usually included in an amount of 0.001% to 50% by weight.

The anti-inflammatory pharmaceutical composition is for preventing and treating various chronic inflammatory diseases such as rheumatoid arthritis, lupus, multiple sclerosis, various acute inflammatory diseases such as sepsis, shock, and rejection of organ transplants, ophthalmic diseases, bronchitis, or inflammatory bowel disease. It can be used for the purpose of.

Examples of the carrier or excipient include water, dextrin, calcium carbonate, lactose, propylene glycol, liquid paraffin, saline, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but are not limited thereto. Two or more carriers or excipients may be used.

In addition, when pharmaceutical compositions for anti-inflammatory drugs, pharmaceutical fillers, extenders, binders, disintegrants, surfactants, anti-agglomerates, lubricants, wetting agents, fragrances, emulsifiers or preservatives may be further included.

In addition, the pharmaceutical composition for anti-inflammatory of the present invention may further include a compound or a plant extract having a known anti-inflammatory activity in addition to the active ingredient, and may be included in 5 to 20 parts by weight, respectively, based on 100 parts by weight of the active ingredient have.

The formulation of the pharmaceutical composition for anti-inflammatory may be in a preferred form depending on the method of use, in particular, a formulation employing a method known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. Can be angry.

Examples of specific formulations include warning agents, granules, lotions, linen agents, limonades, powders, syrups, eye ointments, liquids, aerosols, EXTRACTS, elixirs, ointments, fluid extracts, emulsions, Suspensions, premise, acupuncture, eye drops, tablets, suppositories, injections, spirits, capsules, creams, pills, soft or hard gelatin capsules, and the like.

The pharmaceutical composition for anti-inflammatory according to the present invention may be used orally or parenterally, for example, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural, and oral routes, and its dosage It is good to decide by considering silver, administration method, age, sex and weight of the patient, and severity of disease. For example, the anti-inflammatory pharmaceutical composition of the present invention can be administered once or more at 0.1 mg/kg (body weight) to 100 mg/kg (body weight) per day based on the active ingredient. However, the above dosage is only an example for illustration and is not limited to the above range.

The present invention also provides an anti-inflammatory composition comprising the above-mentioned bovine ultra-high frequency extract as an active ingredient or a cosmetic composition comprising the small-body extract as an active ingredient.

The bovine extract is derived from natural substances, so there are no side effects, there is no cytotoxicity, and because it has an excellent anti-inflammatory effect, it has excellent anti-inflammatory and anti-irritation activities, so that inflammation and externality induced by ingredients included in cosmetics It can effectively control inflammation induced by the environment. Therefore, the anti-inflammatory composition or the bovine ultra-high frequency extract comprising the bovine extract as an active ingredient may be used as an active ingredient of a cosmetic composition having an effect of improving and alleviating inflammation.

The joiner extract may preferably be a joiner microwave extract, more preferably a joiner 70% ethanol aqueous solution microwave extract.

The cosmetic composition may be provided for use as a basic cosmetic, makeup cosmetic, body cosmetic, hair cosmetic, scalp cosmetic, shaving cosmetic, or oral cosmetic.

Examples of the basic cosmetics include cream, lotion, pack, massage cream, latex, etc. Examples of the makeup cosmetics include foundation, makeup base, lipstick, eye shadow, eyeliner, mascara, eyebrow pencil, etc., and the body Examples of cosmetics include soaps, liquid cleaners, bathing agents, sunscreen creams, and sun oils. Examples of the cosmetics for hair are shampoo, conditioner, hair treatment, hair mousse, hair liquid, pomade, hair colorant, and hair bleach Or a color rinse, and examples of the cosmetic for the scalp include hair tonic or scalp treatment, and examples of the shaving cosmetic include aftershave lotion or shaving cream, and examples of the oral cosmetic Toothpaste or mouse washers.

The cosmetic composition, in addition to the active ingredient, the ingredients that are usually formulated in the cosmetic composition according to the use and the nature of the cosmetic composition, for example, moisturizers, ultraviolet absorbers, vitamins, animal and vegetable extracting ingredients, extinguishing agents, whitening agents, vasodilators, astringents, refreshing agents, Hormonal agents and the like may be further added. In addition, the cosmetic composition may further include a base necessary to penetrate or transfer the drug or active ingredient into the skin tissue.

The formulation of the cosmetic composition may take an appropriate form according to the use purpose and the properties of the cosmetic composition, for example, aqueous solution, solubilizing, emulsifying, emulsion, gel, paste, ointment, aerosol, water-oil It may be a two-layer system or a water-oil-powder three-layer system, and examples of the formulation are merely examples, and the formulation and form of the cosmetic composition of the present invention are not limited by the examples.

The active ingredient may be included in 0.001% to 50% by weight based on the total weight of the cosmetic composition, preferably 0.01% to 20% by weight, but the content is an active ingredient contained in the formulation or cosmetic composition It can be appropriately adjusted according to the content of other components, and the content of the active ingredient included in the present invention is not limited by the content.

As a result of confirming through the effect of the bovine microwave extract of the present invention as a plant-derived extract that is a natural product, there is no side effect or safety problem, NO secretion related to inflammation, and the effects of various cytokines on mRNA transcription. It was confirmed that it can effectively inhibit the inflammation. Accordingly, the anti-inflammatory composition comprising the bovine ultra-high extract as an active ingredient is an essential component used in the manufacture of cosmetics in relation to anti-inflammatory or anti-inflammatory pharmaceutical compositions and specific formulations for treating or preventing inflammation-related diseases by inhibiting inflammation. Alternatively, it may be applied to a cosmetic composition having a feature capable of effectively suppressing inflammation of the skin due to the external environment. Therefore, the present invention can be widely used in the field of medical industry related to treatment for diseases related to inflammation and cosmetic industry related to control of skin inflammation.

1 shows an HPLC pattern of 70% ethanol solvent extract (FIG. 1A) and 70% ethanol microwave extract of FIG. 1 (FIG. 1B) according to an embodiment of the present invention. In FIG. 1, CSE-H-70E means 70% ethanol solvent extract, which is a solvent extract obtained by adding 70% ethanol aqueous solution to the wood and performing heat extraction, and CSE-MW-70E is 70% ethanol aqueous solution to the wood. It means a 70% ethanol microwave extract, which is an ultra-high microwave extract added and subjected to microwave extraction.
Figure 2, according to an embodiment of the present invention, 70% ethanol solvent extract of cedar and 70% ethanol microwave extract of cedar NO inhibitory effect (A, D) and PGE ₂ production inhibitory effect (B, E) and iNOS and COX It is a graph showing the inhibitory effect (C,F) of protein expression. In FIG. 2, CSE-H-70E means 70% ethanol solvent extract, which is a solvent extract obtained by adding 70% ethanol aqueous solution to the cattle and performing heat extraction, and CSE-MW-70E is 70% ethanol aqueous solution to the cattle. The ultra-high extract, which is added and subjected to the ultra-high-frequency extraction method, means 70% ethanol ultra-high extract, and the horizontal axis value means the amount added, '+' means addition, and '-' means no addition.
3 is an inflammatory cytokine of IL-1b(A,D), IL-6(B,E) and TNF- of 70% ethanol solvent extract of bovine and 70% ethanol microwave extract of bovine according to an embodiment of the present invention. As a graph showing the effect of inhibiting the production of a(C,F), in FIG. 3, CSE-H-70E means 70% ethanol solvent extract, which is a solvent extract obtained by adding 70% ethanol aqueous solution to the wood and performing heat extraction. And, CSE-MW-70E means 70% ethanol microwave extract, which is a microwave extract obtained by adding 70% ethanol aqueous solution to the cattle and performing microwave extraction, and the horizontal axis value indicates the amount added, and'+' means addition. Means,'-' means no additive
Figure 4 shows the effect of inhibiting the binding activity (binding activity) of NF-κB of 70% ethanol solvent extract (FIG. 4A) and 70% ethanol microwave extract (FIG. 4B) of bovine according to an embodiment of the present invention. As a graph, in FIG. 4A, CSE-H-70E means 70% ethanol solvent extract, which is a solvent extract obtained by adding 70% ethanol aqueous solution to the wood and performing heat extraction, and CSE-MW-70E in FIG. 4B is A 70% ethanol microwave extract, which is a microwave extract obtained by adding 70% ethanol aqueous solution to the cattle and performing microwave extraction, means the amount on the horizontal axis means the amount added, '+' means addition, '-' does not add Means
Figure 5 is a graph confirming the degree of expression of HO-1 protein (FIG. 5a) and mRNA expression of 70% ethanol solvent extract of bovine and 70% ethanol microwave extract of bovine according to an embodiment of the present invention (FIG. 5B) In FIG. 5, CSE-H-70E refers to 70% ethanol solvent extract, which is a solvent extract obtained by adding 70% ethanol aqueous solution to the wood and performing heat extraction, and CSE-MW-70E is 70% ethanol to the wood. Refers to 70% ethanol microwave extract, which is a microwave extract obtained by adding an aqueous solution and performing microwave extraction.
FIG. 6 shows NO production (A), PGE2 production (B), inflammation-mediated cytokine IL-1β (C) by hemeoxygenase-1 protein expression of 70% ethanol microwave extract from bovine according to an embodiment of the present invention. ) And IL-6(D), TNF-α(E), and a graph showing the inhibitory effect of NF-κB binding activity (D). In FIG. 6, CSE-MW-70E means 70% ethanol microwave extract, which is a microwave extract obtained by adding a 70% ethanol aqueous solution to the cattle and performing microwave extraction, and the horizontal axis value represents the added amount, and'+' means It means addition, and'-' means no addition.

Hereinafter, the configuration and effects of the present invention will be described in more detail through specific examples and comparative examples to help understanding of the present invention. However, the following examples are only for understanding the present invention more clearly, and are not limited to the following examples of the present invention, and the protection scope of the present invention should be interpreted by the claims, and is within the equivalent range. All technical ideas should be interpreted as being included in the scope of the present invention.

<Example 1> Preparation of cattle extract

Joiner used in this experiment ( Caesalpinia sappan ) was used to cut the central part of the bark that removed the bark of the stem used as the timber of the timber (Omni Herb Company, Korea), and then used the crushed crushed product.

The joiner extract was prepared by adding a solvent to the lumber stem pulverized product, and then extracting the solvent extract prepared by the solvent extraction method and the microwave extract prepared by the microwave extraction method.

First, a solvent extract was prepared by adding and extracting 70% (v/v) ethanol aqueous solution to the condensate. Specifically, in a 1000 mL round flask, 30 g of the wood flour and 500 mL of a 70% (v/v) ethanol aqueous solution were added and extracted at 80° C. for 15 minutes, filtered with filter paper, the filtrate was concentrated under reduced pressure, and dried. The extraction solvent was removed by the method to obtain 240 mg (yield 0.8%) of a solvent extract (CSE-H-70E) of 70% ethanol aqueous solution.

In addition, for microwave extract, add 30 g of the wood flour and 500 mL of a 70% (v/v) ethanol aqueous solution to a 1000 mL round flask, close the inlet with a lab, and then drill several holes with a diameter of 0.5 cm or less through 2450 Hz and 700 Under the condition of W, microwave extraction was performed for 15 minutes. Specifically, the ultra-short plate extraction was performed for a total of 3 times for 5 minutes, and was performed for 15 minutes. In addition, after performing ultra-short plate extraction for 5 minutes each, using 500 mL of cooling water (water, 4°C) in a 1000 mL beaker, immersed in cooling water for 5 minutes in a flask sieve to cool, and then again performed microwave extraction for 5 minutes. It was carried out in a manner. The cooling water was replaced with another cooling water after performing a cooling process for cooling efficiency. After performing the above three ultra-short plate extractions and proceeding for a total of 15 minutes, the extracted extract was filtered with filter paper, the filtrate was concentrated under reduced pressure, and the extraction solvent was removed by drying to remove 70% ethanol ultra-short plate extract (CSE) -MW-70E) 345 mg (yield 1.15%) was obtained.

<Example 2> HPLC pattern analysis comparison of the extract according to the extraction method

The 70% ethanol solvent extract (CSE-H-70E) and the 70% ethanol microwave extract (CSE-MW-70E) prepared in Example 1 were analyzed by HPLC using patterns of the components. The HPLC apparatus used was YOUNGLIN-YL9100 (Younglin, Anyang, Korea), and the column was a Shiseido Capcell Pak C ₁₈ column (4.6 × 150 mm, 5 μm). As the mobile phase, methanol (MeOH, JT Baker, UK) and water were used, the temperature was performed at 15 to 20, and the flow rate was 0.7 mL per minute. The chromatogram was detected at 254 nm wavelength. As a mobile phase solvent, the composition was changed from 10% (v/v) methanol aqueous solution (methanol (MeOH): water (1:9)) to 100% methanol (MeOH) for 50 minutes. The analyzed results are shown in FIGS. 1A and 1B.

As shown in Figures 1a and 1b, the 70% ethanol microwave extract (CSE-MW-70E) of bovine is specifically compared to the 70% ethanol solvent extract (CSE-H-70E) of bovine 20 It can be seen that the peak is clearly increased. It is evaluated that the increase in the content of the components in the 70% ethanol microwave extract (CSE-MW-70E) of the consignment was marked, and through this, the change of the components in accordance with the extraction method by the solvent extraction method and the microwave extraction method is different from the existing extraction method. Otherwise, it was clear.

< Example 3> Measurement of anti-inflammatory effect according to extraction method-Inhibition effect of inflammatory mediator expression

Generation of NO, PGE ₂ , inflammation mediators in RAW 264.7 cells of 70% ethanol solvent extract (CSE-H-70E) and 70% ethanol microwave extract (CSE-MW-70E) prepared in Example 1 And iNOS, COX-2, the experiment was conducted using the method of Kim et al., European Journal of Pharmacology, 721, pp. 267-276 (2013).

First, RAW 264.7 cells were treated with a 70% ethanol heated extract of bovine and a 70% ethanol microwave extract of bovine at a content of 2.5, 5, 10, and 20 μg/ml, respectively, and pretreated after 3 hours of cultivation, followed by an inflammatory reaction with lipopolysaccharide. After induction, it was cultured in a 5% CO ₂ incubator for 24 hours. After cell culture, NO, PGE ₂ production amount and iNOS, COX-2 protein expression levels were confirmed, and then shown in FIG. 2.

Specifically, in order to confirm the effect of promoting the expression of iNOS and COX-2 proteins according to the content and processing time of the 70% ethanol heated extract and microwave extract of the joining, the experimental method of performing western blot analysis was as follows. Same as

First, after culturing RAW264.7 cells for 12 hours at a concentration of 3 X 10 ⁶ cells/ml in a 60 mm dish, 70% ethanol heating extract and microwave extract of cattle are treated for each concentration, followed by pretreatment for 3 hours. , Lipopolysaccharide was treated with 1 μg/ml and inflammatory reaction was incubated for 24 hours. After the incubation, RIPA (89900, Thermo) buffer was added and centrifuged at 4°C and 14,000 X g for 25 minutes to separate the supernatant. Protein determination in the separated supernatant was performed using a BSA protein experiment kit (Pierce Biotechnology).

Specifically, the supernatant was electrophoresed for 2 hours using a 7.5% SDS-polyacrylamide gel, and then transferred from the polyacrylamide gel using a nitrocellulose membrane (NC membrane). Did. The transferred nitrocellulose membrane was stopped for 1 hour in a fresh blocking buffer (0.1% Tween 20 in Tris-buffered saline) containing 5% fat-free oil. After the reaction was stopped, the membrane was washed three times once every 10 minutes with PBST (PBS, 0.1% Tween 20), followed by iNOS (SC-650, Santacruz biotechnology, USA), COX-2 (SC-1745, Santacruz) The antibody (Ab) of biotechnology, USA) was diluted 1:1000 and reacted for 3 hours. After the reaction, the PBST was washed three times once every 10 minutes, and the secondary antibody (Anti-rabbit IgG, SC-2004, Santacruz biotechnology, USA) was diluted 1:1000 and reacted for 1 hour.

After the reaction, washed three times with PBST once every 10 minutes, and then ECL (Amersham Pharmacia Biotech) solution was mixed well 1:1 and poured onto a nitrocellulose membrane (NC membrane) to emit light, and X-ray in the dark. It developed after photosensitive to the film. The content of Actin was measured using an antibody against Actin (SC-1616, Santacruz biotechnology, USA) in the same manner, and this was quantified based on this. The measured and quantified results are shown in FIG. 2.

As shown in FIG. 2, NO and PGE ₂ production, which were increased when the lipopolysaccharide was treated, was concentration-dependently inhibited when 2.5, 5, 10, and 20 μg/ml of 70% ethanol microwave extract of cattle was treated. The effect was confirmed. This is a result that is significantly compared to the ethanol heated extract showing a significant anti-inflammatory effect only when treated with 20 μg / ml, it was confirmed that microwave extraction can show a significantly superior anti-inflammatory effect compared to solvent extraction.

< Example 4> Measurement of anti-inflammatory effect according to extraction method-Inhibitory effect of inflammatory cytokine production

To confirm the effect of inhibiting inflammatory cytokine production in RAW 264.7 cells of 70% ethanol solvent extract (CSE-H-70E) and 70% ethanol microwave extract (CSE-MW-70E) prepared in Example 1 , RAW 264.7 cells treated with 70% ethanol heated extracts of bovine and 70% ethanol microwave extracts of bovine at 2.5, 5, 10, and 20 μg/ml content, respectively, and pre-incubated for 3 hours, causing inflammatory reaction with lipopolysaccharide Then, incubated in a 5% CO ₂ incubator for 24 hours. After cell culture, the cultured supernatant was used to confirm the production of inflammatory cytokines IL-1β, IL-6, and TNF-α by enzyme immunoassay, and then shown in FIG. 3.

As shown in FIG. 3, IL-1β, IL-6, and TNF-α production increased when treated with lipopolysaccharide was used for 2.5, 5, 10, and 20 μg/ml of 70% ethanol microwave extract from cattle. When it was treated, it was confirmed that the concentration-dependent inhibitory effect was observed at all concentrations. This is a result that does not show an effect until 10 μg/ml is treated, but is significantly compared to an ethanol heated extract that shows a significant anti-inflammatory effect only when 20 μg/ml is treated. It was confirmed that it can exhibit a remarkably superior anti-inflammatory effect.

<Example 5> Measurement of anti-inflammatory effect according to the extraction method-NFκB p65 binding ability

To confirm the effect of 70% ethanol solvent extract (CSE-H-70E) and 70% ethanol microwave extract (CSE-MW-70E) prepared in Example 1 on NFκB-related pathways in RAW 264.7 cells , RAW 264.7 cells treated with 70% ethanol heated extracts of bovine and 70% ethanol microwave extracts of bovine at 2.5, 5, 10, and 20 μg/ml content, respectively, and pre-incubated for 3 hours, causing inflammatory reaction with lipopolysaccharide Then, incubated in a 5% CO ₂ incubator for 24 hours. In order to examine the inhibition of NFκB p65 binding capacity after cell culture, a nuclear separation experiment was conducted using the cultured supernatant using the method of Kim et al., European Journal of Pharmacology, 721, pp. 267-276, (2013). In addition, an experiment was performed to confirm the NFκB p65 binding ability using Western blot analysis, which is the same method as in Example 3, and the results are shown in FIG. 4.

As shown in Figure 4, when the treatment of the 70% ethanol microwave extract of bovine, 2.5, 5, 10 and 20 μg/ml, it was confirmed that the concentration-dependent inhibitory effect at all concentrations, but 70% ethanol heated extract of bovine It was confirmed that only 20 μg/ml showed a significant anti-inflammatory effect, and microwave extraction could exhibit a significantly better anti-inflammatory effect compared to solvent extraction.

< Example 6> Measurement of anti-inflammatory effect by extraction method- Hemeoxygenase -1(HO-1) effect on protein and mRNA expression

To determine the effect on hemeoxygenase (HO-1) protein and mRNA expression in mouse-derived macrophages RAW264.7 cells, 10% and 20 μg/ of 70% ethanol heated extract and 70% ethanol microwave extract, respectively. ml was treated and incubated in 5% CO ₂ incubator for 12 hours. As in Example 3, an experiment was performed to confirm the amount of HO-1 protein by western blot analysis, and the results are shown in FIG. 5A, and the amount of HO-1 mRNA expression through RT-PCR technique. By performing an experiment to confirm, the results are shown in Figure 5b.

As shown in FIG. 5, in the case of 70% ethanol microwave extract of the bovine, the expression of HO-1 increased (FIG. 5A), while the RT-PCR experiment HO-1 mRNA production also increased (FIG. 5B), 70% of the bovine. Ethanol In the case of the ethanol heated extract, there was no significant increase, and it was confirmed that the anti-inflammatory effect of the 70% ethanol microwave extract of wood was remarkably excellent.

<Example 7> Inflammatory response suppression effect through expression of inflammation-related protein

Inhibition of NO production through hemeoxygenase-1 (HO-1) protein expression in mouse derived macrophages RAW 264.7 cells, inhibition of PGE2 production, inflammatory mediator cytokines IL-1β, IL-6, TNF-α and NFκB p65 The effect on the inhibition of binding capacity was confirmed.

The primary macrophages isolated from the abdominal cavity of the mouse were treated with 50 μM of hemoxygenase-1 inhibitor SnPP (Tin protoporphyrin, Porphyrin Products, Logan, UT) 3 hours before, followed by 2.5, 5, and 70% ethanol microwave extracts from cattle. Treatment was performed at a concentration of 10 and 20 μg/ml for 12 hours, and an inflammatory reaction was induced with LPS, followed by incubation in a 5% CO ₂ incubator for 24 hours. NO production inhibition (6A) and PGE ₂ production inhibition (6B) and inflammatory mediator cytokines IL-1β(6C), IL-6(6D), and TNF-α(6E) through hemeoxygenase-1 protein expression And NFκB p65 binding capacity (6F) inhibitory effect is shown in FIG. 6.

As shown in FIG. 6, the 70% ethanol microwave extract of bovine showed a remarkable effect of inhibiting NO, PGE2, IL-1β, IL-6, TNF-α production and NFκB p65 binding capacity, which were increased during LPS treatment. However, as a result of pre-treatment with SnPP, an inhibitor of hemeoxygenase-1, it was confirmed that decreased NO, PGE ₂ , IL-1β, IL-6, TNF-α production and NFκB p65 binding capacity increased. It has an effect of inhibiting NO(6A), PGE2(6B), IL-1β(6C), IL-6(6D), TNF-α(6E), and NFκB p65 binding capacity (6F) of the 70% ethanol microwave extract of cattle. It is a result of confirming that it is due to the expression of sigenase-1.

Thus, the 70% ethanol microwave extract of bovine wood is an inflammation induced in macrophages by increasing the expression and activity of hemooxygenase-1, unlike the 70% ethanol heated extract of bovine, which does not affect the expression and activity of hemeoxygenase-1 It was confirmed that the inhibitory effect of the reaction was excellent.

According to the results of the above experiment, the extract of bovine that can be used as an edible product is a microwave extraction method compared to the existing solvent extraction method, considering the results related to the protein causing inflammation and various cytokines causing inflammation in relation to the anti-inflammatory effect. It was confirmed that the prepared extract can inhibit inflammation very effectively, and it was confirmed that performing with the conditions of 2450 Hz with respect to the bovine microwave extract, has excellent anti-inflammatory effect compared to other conditions, the bovine extract of the present invention In particular, 70% ethanol aqueous solution ultra-short extracts of pine, preferably 70% ethanol aqueous solution ultra-high extracts of pine produced under conditions of 2450 Hz and 700 W can not only replace the existing inflammatory treatment, but also inflammation caused by components used in existing cosmetics It is expected that the occurrence can be appropriately controlled and used as an active ingredient in the cosmetic composition.

Claims (8)

Anti-inflammatory pharmaceutical composition comprising 70% (v/v) ethanol aqueous solution in the cedar ( Caesalpinia sappan ) stem and extracting the cedar microwave extract extracted by microwave extraction under the conditions of 2,450 Hz as an active ingredient. delete delete delete A cosmetic composition having an anti-inflammatory effect comprising 70% (v/v) ethanol aqueous solution in the stem of Caesalpinia sappan and extracting the microwave extract extracted by microwave extraction at 2,450 Hz as an active ingredient. delete delete A joiner preparation step of preparing a joiner stem crushed product by drying and crushing the joiner stem; And adding 70% (v/v) ethanol aqueous solution to the trunk stem pulverized material, performing microwave extraction at 2,450 Hz for 5 minutes, and then repeating the process of cooling in 4° C. cooling water for 5 minutes for 3 minutes to total 15 minutes. During the extraction process during the extraction process 70% (v/v) ethanol aqueous solution microwave extract extract step
A method of manufacturing bovine ultra-high wave extract having an anti-inflammatory effect, comprising a.

Images