KR101780939B1 - Method for Seperation of Compound Derived from Ginseng and Composition for anti-inflammatory Using the same - Google Patents

Abstract

The present invention relates to a ginseng-based compound separation method and an anti-inflammatory composition using the same, and the separation method of the present invention can effectively isolate compounds having anti-inflammatory activity other than ginsenoside-based compounds in ginseng extract, The isolated compounds are more harmless to the human body than the conventional synthetic pharmaceutical compositions, have no side effects, and can effectively inhibit the generation of inflammatory mediators. Therefore, the pharmaceutical composition or the composition for external application for skin can be used for preventing or treating inflammatory diseases, The branch can be used for various products.

Description

TECHNICAL FIELD The present invention relates to a method for separating ginseng-derived compounds and an antiinflammatory composition using the same,

The present invention relates to a method for separating ginseng-derived compounds and an anti-inflammatory composition using the same. More particularly, the present invention relates to a method for separating a compound having anti-inflammatory activity from a ginseng leaf extract and an antiinflammatory composition comprising the separated compound as an active ingredient will be. The compound effectively inhibits the production of inflammatory mediators and has little toxicity, and thus can be used as a pharmaceutical composition for preventing or treating inflammatory diseases or a composition for external application for skin.

The inflammatory reaction is one of the damage of the tissue, the external stimulus, or the defense of the biotissue to various infectious agents. The inflammatory reaction is caused by enzymatic activation, inflammatory mediator secretion, cell mediated by various inflammatory mediators in blood vessels and body fluids, Infiltration and fluid exudation, circulatory disturbances, tissue degeneration and hyperplasia. During the inflammatory process, the macrophages accumulate at the wound site in the early stage, attack the invading bacteria, accumulate plasma at the wound area, and increase the blood flow, resulting in external symptoms such as fever, erythema, edema, and pain. If the inflammatory reaction continues or occurs excessively, it progresses to a major pathological condition (irritable allergic disease, chronic inflammatory disease) of the disease, resulting in serious abnormal disorder.

Nonsteroidal antiinflammatory drugs (NSAIDS), a widely used agent for the treatment of most inflammatory diseases, are produced from arachidonic acid called cyclooxygenase (COX), prostaglandin biosynthesis Inhibition of the enzyme activity involved in the treatment of gastrointestinal disorders, it exhibits anti-inflammatory action. In addition to its main therapeutic effect, it causes severe side effects such as gastrointestinal disorders, hepatic disorders, renal diseases and the like. Therefore, it is widely demanded to develop a new anti-inflammatory analgesic agent which is excellent in anti-inflammatory efficacy while having no side effects and being usable for a long time.

Various biochemical phenomena are involved in the cause of inflammation in living body. Macrophage is a multifunctional cell that produces various cytokines and nitric oxide (NO) by chemical stimulation and plays an important role in the inflammatory response. As a result of the inflammatory response, the above-mentioned supraphysiological concentrations of nitric oxide produced by iNOS (inducible nitric oxide synthase) play a role in the pathology of various inflammatory diseases. Among the inducers of inflammation, LPS (lipopolysaccharide) interacts with immune cells such as leukocytes, and it is important in the inflammatory response by the increase of nitric oxide concentration by activation of iNOS isoform in monocyte macrophages It plays a role. LPS is an endotoxin present in the outer membrane of Gram-negative bacteria. Binding to TLR4 (tolllike receptor 4) results in activation of many cytokine genes such as IL-6 (interleukin-6) and chemokines Initiating signal transduction leading to inflammation in that area. In particular, inducible nitric oxide synthase (iNOS) expressed by cytokine stimulation such as interferon gamma and TNF-alpha produces a large amount of nitrogen oxides (NO) over a long period of time. These oxidative stresses are known to promote NFκB activity, a transcription factor of inflammatory responses inhibited by IκB. Activated NFκB is known to migrate to the nucleus and promote the expression of genes that induce inflammatory responses such as iNOS, IL-1β, and TNF-α. Various inhibitors of these factors inhibit the inflammatory response It is known.

According to a recent study, NO is a free radical generated from arginine by three major NOS isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). nNOS and eNOS are controlled by Ca ^{2 +} / calmodulin (calmodulin), but, iNOS is regulated at the transcriptional level by inflammatory stimuli such as IL (interleukin), IFN (interferon), LPS. NO produced in a few seconds by nNOS or eNOS is responsible for normal physiological functions such as vasodilation, neurotransmission, cell destruction to pathogen, etc. However, overexpressed NO by iNOS in macrophages reacts with superoxide (Peroxynitrite), which acts as a powerful oxidant, damages cells and activates NFκB, a transcription factor in macrophages activated by inflammatory stimuli, in response to chronic diseases such as inflammation, cancer, and arteriosclerosis It is known to be related.

Therefore, the development of a compound that inhibits NO produced is highly likely to be developed as a therapeutic agent for the above diseases, and from this viewpoint, a compound that inhibits NO production can be used as a therapeutic agent for various inflammatory diseases of the human body.

In recent years, there has been an increasing demand for pharmaceuticals using natural products in accordance with naturalism and well-being trends. To solve this problem, a variety of methods for producing extracts have been developed, and functional foods and medicines using natural extracts have been developed. However, development of a natural pharmaceutical composition or a raw material thereof having less side effects than a conventional synthetic pharmaceutical composition while still having an excellent inflammatory disease therapeutic effect is insufficient.

Ginseng (Panax ginseng C.A. Meyer) is a herb that has been used in Korea, China and Japan for over 2,000 years, and has been used for the purpose of preventing disease and prolonging the life span. The efficacy and effect of ginseng which has been known so far can be determined by the action of the central nervous system, the anticarcinogenic action, the anticancer activity, the immune function regulating action, the anti-diabetic action, the hyperfunction of liver function, the improvement of cardiovascular disorder, (Korean Ginseng & Tobacco Research Institute, 56-112, 1996). In addition, it has been reported that the antioxidant activity and antioxidant activity of osteoporosis are improved.

Ginsenoside, which is a representative physiologically active ingredient of ginseng, is distributed evenly on the ground and underground of ginseng. Especially, ginsenoside (root), ginseng leaf and ginseng berries have not only ginsenoside content but also composition (Attele AS et al . , Biochem . Pharmacol. , 58; 1685-1693, 1999).

In the prior art, various ginsenosides derived from ginseng have been reported to have antioxidant, anti-inflammatory, whitening and moisturizing effects (Korea Patent Publication No. 2014-0006418; Korean Patent Publication No. 2013-0031988) The effects of ginseng extracts on the anti - inflammatory effects of ginseng extracts have been limited.

Accordingly, the present inventors have made intensive efforts to select substances having anti-inflammatory activity and no toxicity, and as a result, compounds other than ginsenosides were extracted and isolated from ginseng leaves. The isolated compound is cytotoxic and inhibits the generation of nitric oxide (NO); And tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 Inhibiting the inflammation through at least one mechanism selected from the group consisting of inhibiting the production of one or more inflammatory cytokines, and completed the present invention.

It is an object of the present invention to provide a method for separating ginseng-derived compounds having anti-inflammatory activity.

The present invention also provides a pharmaceutical composition for preventing or treating an inflammatory disease comprising the compound, a skin external preparation containing the composition, or a cosmetic composition for improving inflammatory diseases.

In order to accomplish the above object, the present invention provides a method for producing a ginseng extract, comprising: obtaining an extract from ginseng; Obtaining a liquid distribution extract using an organic solvent with an extract which is concentrated under reduced pressure; And separating and purifying the liquid distribution extract to obtain a compound represented by the following formula (1).

[Chemical Formula 1]

Wherein R1 and R2 are, each independently, a carboxyl group (carboxylic group) of C12 to C25, including a hydrogen atom, or 0-5 double bonds, however, R ₁ And R ₂ are both hydrogen atoms are excluded.

According to a preferred embodiment of the present invention, the ginseng may be a ginseng leaf.

According to another preferred embodiment of the present invention, the ginseng may be ginseng obtained by multistage cultivation, hydroponic cultivation or organic cultivation.

According to another preferred embodiment of the present invention, the ginseng extract is water, C1-C5 And may be extracted with at least one solvent selected from the group consisting of alcohol, ethyl acetate, chloroform, butanol, hexane, and mixtures thereof.

According to another preferred embodiment of the present invention, the organic solvent is water, C1-C5 Alcohol, ethyl acetate, chloroform, butanol, hexane, and mixtures thereof.

According to another preferred embodiment of the present invention, the liquid distribution extract is obtained by dividing and extracting ginseng extract concentrated under reduced pressure using ethyl acetate (EtOAc) / water (H ₂ O); Obtaining and dispersing the water layer with butanol (n-buOH); Concentrating the water layer and the butanol layer under reduced pressure to obtain ethyl acetate, butanol and water fractions; And a step of obtaining the ethyl acetate layer.

According to another preferred embodiment of the present invention, the formula (1) may be represented by the following formulas (2) to (4).

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

According to another preferred embodiment of the present invention, the compound may have anti-inflammatory activity and inhibit the generation of nitric oxide (NO); And tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 And inhibiting the production of any one or more inflammatory cytokines. The inflammation can be suppressed through at least one mechanism selected from the group consisting of.

The present invention also relates to a compound isolated by the above method; And a salt thereof, as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating an inflammatory disease.

According to a preferred embodiment of the present invention, the inflammatory disease may be at least one selected from the group consisting of dermatitis, sore throat, tonsillitis, fibromyalgia, rheumatoid arthritis, shoulder periitis, myositis, hepatitis, cystitis and nephritis.

The present invention also provides an external preparation for skin comprising the composition.

According to a preferred embodiment of the present invention, the external preparation for skin may be for prevention or treatment of atopy.

The present invention also relates to a compound isolated by the above method; And a salt thereof, as an active ingredient. The present invention provides a cosmetic composition for preventing or ameliorating an inflammatory disease.

The present invention also relates to a compound isolated by the above method; And salts thereof; and a quasi-drug composition for preventing or ameliorating an inflammatory disease comprising at least one member selected from the group consisting of:

According to one preferred embodiment of the present invention, the quasi-drug composition is any one of the group consisting of disinfectant cleaner, shower foam, gagrin, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment agent, coating agent, .

The separation method of the present invention can effectively isolate compounds having antiinflammatory activity other than the ginsenoside compounds in the ginseng extract and the compounds isolated by the above method are harmless to the human body and have no side effects, Inflammatory mediators can be effectively inhibited. Therefore, the present invention can be applied not only to pharmaceutical compositions for preventing or treating inflammatory diseases or compositions for external application for skin, but also to various products having anti-inflammatory activity.

Brief Description of the Drawings Fig. 1 is data obtained by confirming the nitric oxide (A) and cell viability (B) according to the treatment concentration of the compound represented by the formula (1) of the present invention.
2 is a graph showing tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-1 beta (IL-6: interleukin-6).

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

As described above, development of a pharmaceutical composition or a raw material thereof which has anti-inflammatory effect and has fewer side effects than conventional synthetic pharmaceutical compositions has been insufficient.

The present invention solves the above-mentioned problems by providing a method for separating a compound having anti-inflammatory activity derived from ginseng. Thus, compounds which are more harmless to the human body than the conventional synthetic whitening ingredients and have little side effects and can be effectively used for long-term use with little human side effects and can effectively inhibit the generation of inflammatory mediators are provided, and the prevention of inflammatory diseases Or a pharmaceutical composition for treatment or an external composition for skin.

Accordingly, the present invention relates to a method for producing a ginseng extract, comprising: obtaining an extract from ginseng and concentrating it under reduced pressure;

Obtaining a liquid distribution extract using an organic solvent with an extract which is concentrated under reduced pressure; And

And separating and purifying the liquid distribution extract to obtain a compound represented by the following formula (1).

[Chemical Formula 1]

Wherein R1 and R2 are, each independently, a carboxyl group (carboxylic group) of C12 to C25, including a hydrogen atom, or 0-5 double bonds, however, R ₁ And R ₂ are both hydrogen atoms are excluded.

The carboxyl group may preferably be represented by the following formula (6): wherein n is 2 to 5 and R ₃ is a straight-chain alkyl group of C7 to C18 or a C7 to C18 Lt; / RTI >

[Chemical Formula 6]

The carboxyl group may be more preferably represented by the following formulas (7) to (12).

(7)

[Chemical Formula 8]

[Chemical Formula 9]

[Chemical formula 10]

(11)

[Chemical Formula 12]

Further, the formula (1) of the present invention may most preferably be represented by the following formulas (2) to (4).

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

When the compound of Formula 1 of the present invention is extracted from ginseng, it can be preferably isolated and purified from ginseng leaf extract. The ginseng may be any ginseng regardless of the variety and cultivation method. Preferably, ginseng obtained by multistage cultivation, hydroponic cultivation or organic cultivation can be used, and more preferably, hydroponically cultivated ginseng can be used.

Hereinafter, the separation method of the present invention will be described in detail.

First, the step of obtaining an extract from ginseng leaves and concentrating under reduced pressure will be described.

The extraction can be carried out using an extraction solvent. The extraction solvent is not particularly limited as long as it is ordinarily used for extracting natural products, but it may preferably be any one or more selected from C1 to C5 alcohols, more preferably methanol Or ethanol.

Further, the method for obtaining the above extract is not particularly limited as long as it is a method for obtaining an extract from a natural product, but it is preferable to use at least one selected from the group consisting of ultrasonic extraction, filtration and reflux extraction, It is preferable to use a vacuum decompressing concentrator or a vacuum rotary evaporator, but the present invention is not limited thereto.

Next, the step of obtaining a liquid distribution extract by using an organic solvent as an extract obtained by concentration under reduced pressure is described.

The organic solvent is not particularly limited as long as it is an organic solvent usually used for extraction, but it may preferably be at least one selected from the group consisting of C1 to C5 alcohol, water, hexane, methylene chloride and ethyl acetate, May comprise water and / or ethyl acetate, and most preferably ethyl acetate may be used.

The liquid distribution extract is not particularly limited as long as it is prepared using a conventional liquid distribution extraction method. The liquid dispensing and extraction method is carried out in order to remove a large amount of impurities from a polar substance to a non-polar substance contained in the extract to perform purification using the next step chromatography more effectively. And can be removed by using the solubility and the miscibility of the solvent and the solvent.

Finally, the step of obtaining the fractions by chromatography of the liquid distribution extract is described.

The chromatography is not particularly limited as long as it is a chromatographic method usually used for component separation. For example, C ₁₈ column chromatography (C ₁₈ column chromatography, SiO ₂ column chromatography, thin-layer chromatography, or high performance liquid chromatography.

The chromatography is not particularly limited as long as it is a condition usually used for separating the respective components. Chromatography can be carried out once to several times, preferably two times or more, more preferably three times or more. When the multiple chromatography as described above is carried out, the purity of the obtained fractions can be increased to obtain fractions with high purity.

In one embodiment of the present invention, preferably, the freeze-dried ginseng leaves are extracted by immersing in methanol and then concentrated under reduced pressure to obtain an extract; The ginseng leaf methanol extract is separated and extracted with ethyl acetate (EtOAc) / water (H ₂ O), and then the aqueous layer is separated and extracted with butanol (n-buOH) Concentrating the water layer and the butanol layer under reduced pressure to obtain ethyl acetate, butanol and water fractions; And separating and purifying by silica gel column chromatography and reverse phase silica gel column chromatography to obtain a compound represented by formula (1).

In the present invention, the following four compounds represented by the following formulas (2) to (5) were obtained by the above method, and they were respectively classified into panaxcerol A (Formula 2), panaxcerol B C (panaxcerol C, formula 4) and panaxcerol D (formula 5).

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

The compound represented by Formula 1 is characterized by having anti-inflammatory activity,

Inhibiting the production of nitric oxide (NO); And

(TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) Inhibiting the production of one or more inflammatory cytokines;

Lt; RTI ID = 0.0 > and / or < / RTI >

In one embodiment of the present invention, BV2 cells are stimulated with LPS to confirm the anti-inflammatory effect of each of the flushing roll A, flushing roll B, flushing roll C and flushing roll D, so that inflammation mediated by LPS stimulation The inhibitory effect on the production of sex steroids and cytotoxicity were observed.

 Lipopolysaccharide (LPS), which is well-known as an endotoxin, is the most well known external factor involved in the activation of macrophages. In particular, TNF-α (TNF-α), IL-6 (pro-inflammatory cytokines) such as IL-1β (interleukin-6) and IL-1β (interleukin-1 beta) In addition, when LPS stimulates macrophages, NO is produced from macrophages by the production of nitrogen monoxide (NO) in the process of converting L-arginine into L-citrulline by an enzyme called iNOS (inducible nitric oxide synthase). In mammals, NO is synthesized by three types of NO synthase (NOS): nNOS (neuronal NOS), eNOS (endothelial NOS) and iNOS (inducible NOS). Among these, NO produced by nNOS and eNOS is produced for normal biological function, and concentration in tissues is kept low to a certain level. However, NO produced by iNOS is overexpressed and exhibits deleterious effects on living organisms such as pathological vasodilation, cytotoxicity, and tissue damage.

As shown in Fig. 1A and Table 1, both the flounder roll A to flounder roll D showed inhibitory activity on the production of nitrogen monoxide (NO), one of the inflammation inducers of inflammatory diseases, in a concentration-dependent manner, and inhibited 50% , The IC ₅₀ values were found to be 63.8 μM, 59.4 μM, 7.7 μM and 8.0 μM for the ripened roll A, ripened roll B, ripened roll C and ripened roll D, respectively.

In the case of cytotoxicity, as shown in Fig. 1B, no cytotoxicity was observed even at a concentration of 100 [mu] M or higher in the strike roll A and strike roll B, and the strike roll C and strike roll D were slightly Was observed.

However, it has been confirmed that the IC ₅₀ values of the above compounds are all in a concentration range that does not show cytotoxicity and can be used without toxicity within an effective range.

Figure 2 is a graph showing tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) -6), indicating that the expression of IL-1β, IL-6, and TNF-α mRNA was reduced in a concentration-dependent manner in all four compounds.

That is, the compound of Formula 1 of the present invention not only has a whitening activity through inhibition of melanin biosynthesis, but also inhibits the production of nitric oxide which is an inflammatory mediator; And tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 Inhibition of inflammation through at least one mechanism selected from the group consisting of inhibiting the production of any one or more inflammatory cytokines.

Further, the present invention relates to a compound separated by the above method; And salts thereof; and a pharmaceutical composition for the prophylaxis or treatment of inflammatory diseases comprising at least one member selected from the group consisting of:

The salt of the compound represented by the formula (1) may be an acid addition salt formed by a pharmaceutically acceptable free acid, and the free acid may be an organic acid or an inorganic acid. Examples of the inorganic acid include hydrochloric acid, Sulfuric acid, sulfurous acid and phosphoric acid can be used. Examples of the organic acid include citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, metal sulfonic acid, acetic acid, glycolic acid, succinic acid, tartaric acid, Acetic acid, lactic acid, malonic acid, glutamic acid, citric acid, and spartanic acid.

The addition salt according to the present invention can be obtained by a conventional method, that is, dissolving the compound represented by Formula 1 in a water-miscible organic solvent such as acetone, methanol, ethanol or acetonitrile, adding an equivalent amount or an excess amount of an organic acid, Or by precipitation or crystallization after adding an aqueous solution, or by evaporating a solvent or an excessive amount of acid, and then drying or precipitating the salt by suction filtration.

The present invention includes not only compounds represented by the above formula (1) and pharmaceutically acceptable salts thereof, but also solvates, hydrates and stereoisomers which exhibit the same effects as those which can be prepared therefrom.

Furthermore, the pharmaceutically acceptable salts thereof are lyophilized and include such agents or salts thereof that can be reconstituted to form pharmaceutically acceptable agents for administration, such as by intravenous, intramuscular or subcutaneous injection .

In addition, the compounds of formula (I) as described herein or their pharmaceutically acceptable salts can be administered orally, solid or as a solid, or intramuscularly or intravenously as a solution, suspension or emulsion. Preferably as a liposomal suspension, intravenously or intramuscularly, and may be provided with a pharmaceutical formulation suitable for administration by inhalation as an aerosol.

The pharmaceutical composition may be used to prevent or treat an inflammatory disease.

The inflammatory disease is not limited as long as it is a disease associated with an inflammatory reaction and includes, for example, rheumatoid arthritis, arteriosclerosis, dermatitis, edema, systemic lupus erythematosis, scleroderma, ulcerative colitis, psoriasis, anaphylactic, Retinopathy, retinitis, macular degeneration, uveitis, conjunctivitis, arthritis, ankylosing spondylitis, osteoarthritis, osteoporosis, allergy, diabetes, diabetic nephropathy, nephritis, nephritis, Sjogren's syndrome, Crohn's disease, autoimmune pancreatitis, periodontal disease, asthma, graft versus host Chronic pelvic inflammatory disease, endometritis, rhinitis, tonsillitis, otitis media, sore throat, cystitis, fibromyalgia, shoulder circumference, myositis, hepatitis or chronic prostatitis. Preferably atopic dermatitis, sore throat, tonsillitis, fibromyalgia, rheumatoid arthritis, shoulder periitis, myositis, hepatitis, cystitis or nephritis.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. The pharmaceutical composition containing a pharmaceutically acceptable carrier may be formulated into tablets or capsules by oral administration in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories, Can be used.

More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like. Such solid preparations can be prepared by incorporating at least one excipient into the compound of formula (I) or a pharmaceutically acceptable salt thereof, Starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used.

Examples of liquid formulations for oral use include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition for the prevention or treatment of inflammatory diseases according to the present invention is administered in a pharmaceutically effective amount. The "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the species and severity, age, sex, drug activity, The time of administration, the route of administration and the rate of excretion, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by a person skilled in the art.

In addition, the pharmaceutical composition may be used alone or in combination with methods using surgery, hormone therapy, drug therapy, and biological response modifiers for the prevention or treatment of inflammatory diseases.

Further, the pharmaceutical composition may be administered in combination with a conventional therapeutic agent for inflammatory diseases. The administration route of the composition may be administered through any conventional route as long as it can reach the target tissue. But are not limited to, intravenous, intravenous, intramuscular, subcutaneous, intradermal, oral, intranasal, intrapulmonary, intrathecal.

The present invention also provides an external preparation for skin comprising a pharmaceutical composition for the prevention or treatment of inflammatory diseases as described above.

The formulation of the external preparation for skin is not particularly limited. For example, it can have formulations such as powders, gels, ointments, creams, liquids, aerosols and the like.

In addition, another applicable form of the external preparation for skin of the present invention is a pharmaceutical topical preparation. The external preparation for skin of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the compound represented by the formula (1), which is an active ingredient, in order to have an inflammation-improving effect, and may be manufactured by a topical pharmaceutical preparation. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, , And other conventional additives such as a bacteriostatic agent may be added. It may also be formulated into topical preparations such as ointments, creams, gels, skin emulsions, skin suspensions, patches or sprays according to a conventional method by additionally adding a diluent, a dispersant, a surfactant, a binder and a lubricant. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the methods disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.

Further, the dosage of the external preparation for skin according to the present invention may vary depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease. The daily dose of the compound represented by Formula 1 may be 0.01 to 300 mg / cm 2, and may be continuously applied for 1 month or more after 1 to 5 times of application.

In addition, the inflammatory disease is not limited as long as it is a disease associated with an inflammatory reaction, but may be preferably atopic dermatitis.

Further, the present invention relates to a compound separated by the above method; And a salt thereof; and a cosmetic composition for preventing or improving an inflammatory disease.

The cosmetic composition may be prepared in the form of a general emulsified formulation and a solubilized formulation. Examples of the emulsified formulations include nutritive lotions, creams, essences, and the like, and the solubilization formulations include softening longevity. Suitable formulations include, but are not limited to, solutions, gels, solid or paste anhydrous products, emulsions obtained by dispersing the oil phase in water, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) A cream, a skin, a lotion, a powder, an ointment, a spray, or a conical stick. It may also be in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

Furthermore, the cosmetic composition is not particularly limited as long as it is usually added to cosmetics. For example, it is possible to use other additives such as fatty substances, organic solvents, solubilizers, thickening and gelling agents, softening agents, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, Chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles and the like.

Further, the present invention relates to a compound separated by the above method; And a salt thereof; and a quasi-drug composition for preventing or ameliorating an inflammatory disease, which comprises at least one member selected from the group consisting of:

The term "quasi-quasi-product" means any textile, rubber product or the like used for the purpose of treating, alleviating, treating or preventing a disease of a person or animal, weak acting on the human body or acting directly on the human body, And similar products, products for sterilization, insecticides and similar uses for the prevention of infectious diseases, products for the purpose of diagnosis, treatment, alleviation, treatment or prevention of diseases of human beings or animals Machinery, or apparatus, and that is not an apparatus, machine or apparatus of an article used for the purpose of giving pharmacological effects to the structure or function of a person or animal.

When the quasi-drug composition of the present invention is used as a quasi-drug additive, the quasi drug composition may be added as it is or may be used together with other quasi-drug or quasi-drug components, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.

The quasi-drug composition of the present invention is not limited to this, but it is preferably one selected from the group consisting of disinfectant cleaner, shower foam, gagrin, wet tissue, detergent soap, hand wash, patch, humidifier filler, mask, ointment agent and filter filler Lt; / RTI >

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Compound separation

1-1: Preparation of ginseng leaf extract

After 90 days of harvesting, 6.2 kg of hydroponically grown ginseng leaves were dried in a freeze dryer to obtain a ginseng leaf sample. The ginseng leaf sample was immersed in an aqueous solution of 80% methanol for 24 hours at 25 ° C, and the extract was filtered to obtain a filtrate, which was concentrated under reduced pressure to obtain a concentrated extract of reduced pressure 1 (1.06 kg). Thereafter, the above extraction was repeated twice, and the filtrate obtained by repeated extraction was combined and concentrated under reduced pressure to obtain a reduced pressure concentrated extract 2 (382 g).

The depressurized concentrated extracts 1 and 2 were combined to obtain a depressurized concentrated extract of 1.44 kg in total.

1-2: Ginseng leaves Fraction  Preparation and compound separation

The concentrated extract under reduced pressure obtained in Example 1-1 was subjected to partitional extraction four times with ethyl acetate (EtOAc, 3 L) and water (3 L), and the water layer was again extracted with butanol (n-BuOH, 2.7 L) Lt; / RTI > Each layer (ethyl acetate, water, butanol) was concentrated under reduced pressure to obtain an ethyl acetate distribution extract (75 g), a butanol distribution extract (470 g) and a water distribution extract (855 g).

Example 1-2 silicon dioxide column chromatography to give the ethyl acetate extract obtained in the distribution (SiO ₂ column chromatography, hereinafter referred to as _{'SiO 2 cc') (φ} 14 × 16 ㎝, CHCl 3 -MeOH = 30: 1, 60 ℓ → CHCl ₃ -MeOH-H ₂ O = 15: 3: 1, 136 ℓ), and 150 ml of each solution was collected to obtain an aliquot. The aliquots were confirmed by TLC (CHCl ₃ -MeOH-H ₂ O = 25 L: 3 L: 1 L, 13 L: 3 L: 1 L, 7 L: 3 L: 1 L), and similar portions were collected and concentrated using a vacuum concentrator 24 fractions (HPE 1 to HPE 24) were obtained.

Then, the 24 fractions were used to separate and purify the compound.

(1) Octadecylsilanized silica column chromatography (ODS cc, manufactured by Waters Co.) was applied to the 15th fraction (HPE 15, 5.49 g; Ve / Vt = 0.34-0.36) ; Φ 4 × 9 ㎝, MeOH -H 2 O = 3: 2, 1.0ℓ → 2: 1, 2.5ℓ → 3: 1, 5.2ℓ → 5: 1, 2.0ℓ) carried by a total of 25 fractions (HPE 15-1 to HPE 15-25).

Octadecylsilanized silica column chromatography (Φ 2.5 × 7 cm, MeOH-H ₂ O = 0.34-0.40) was performed on the twelfth fraction (HPE 15-12, 135.2 mg; Ve / Vt = 0.34-0.40) (HPE 15-12-6, 29.4 mg; < RTI ID = 0.0 > V) < / RTI > / Vt = 0.14-0.28) final compound 1 (compound 1) was _{[TLC R f = 0.30 (RP} -18 f 254S, MeOH-H 2 O = 4 obtained _{from: 1), R f = 0.50} (Kieselgel 60 F ₂₅₄ , CHCl ₃ -MeOH-H ₂ O = 10: 3: 1).

(2) Next, octadecylsilane-leased silica column chromatography (Φ 3 × 8 cm, pH 8.0) was performed on the 18th fraction (HPE 15-18, 142.7 mg; Ve / Vt = 0.50-0.58) A total of 7 fractions (HPE 15-18-1 to HPE 15-18-7) were obtained by carrying out the first fraction (HPE 15-18-MeOH-H ₂ O = 4: 1, 900 ml) (TLC R _f = 0.50 (RP-18 F _254S , MeOH-H ₂ O = 6: 1), R _f = 0.40 (Kieselgel 60 F ₂₅₄ , CHCl ₃ -MeOH-H ₂ O = 10: 3: 1).

(3) To the ninth fraction (HPE 9, 9.28 g; Ve / Vt = 0.10-0.16) of the first 24 fractions, SiO ₂ cc (Φ 7 × 15 cm, n-hexane-EtOAc = 1: ), A total of 13 fractions (HPE 9-1 to HPE 9-13) were obtained. For the tenth fraction (HPE 9-10, 4.47 g; Ve / Vt = 0.24-0.98) among the above 13 fractions Octadecylsilanediol column chromatography (Φ 4 × 9 cm, MeOH-H ₂ O = 15: 1, 4 L) was performed to obtain a total of 9 fractions (HPE 9-10-1 to HPE 9-10-9 ).

Octadecylsilanized silica column chromatography (Φ 4 × 6 cm, acetone-hexane) was further performed on the second fraction (HPE 9-10-2, 3.14 g; Ve / Vt = 0.06-0.14) A total of 10 fractions (HPE 9-10-2-1 to HPE 9-10-2-10) were obtained by performing a reaction of acetonitrile-H ₂ O = 2: 2: 1, 3.6 L, Finally, Compound 3 was obtained in the first fraction (HPE 9-10-2-9, 446.0 mg; Ve / Vt = 0.38-0.55) [TLC R _f = 0.50 (RP- 18 F _254S , acetone- acetonitrile-H ₂ O = 7: 3: 1), R _f = 0.55 (Kieselgel 60 F ₂₅₄ , CH ₂ Cl ₂ -MeOH = 10: 1).

(4) The fourth fraction (HPE 9-10-4, 141.6 mg; Ve / Vt) of the nine fractions (HPE 9-10-1 to HPE 9-10-9) = 0.24-0.29) to give compound 4 (TLC R _f = 0.25 (RP-18 F _254S , MeOH-H ₂ O = 50: 1), R _f = 0.50 (Kieselgel 60 F ₂₅₄ , n-hexane-EtOAc = 1: 30)].

Compound structure analysis

Structural analysis for the compound obtained in Example 1 was carried out.

Optical rotations were measured using a JASCO P-1010 digital polarimeter (JASCO P-1010 digital polarimeter; Jasco, Japan) and gas chromatography (GC / MS) using a Shimadzu GCMS-QP2010 Plus mass spectrometer (Shimadzu GCMS-QP2010 Plus mass spectrometer; Shimadzu, Japan).

The Fast atom bombardment spectrum (FAB) / MS) was recorded using a spectrometer (JMS-700, JEOL, Japan). Infrared spectroscopy (IR spectra) was measured using a PerkinElmer spectrum one Fourier transform-IR spectrometer (PerkinElmer, UK).

NMR analysis was carried out by using a 400 MHz FT-NMR spectrometer (Varian Inova AS 400, Palo Alto, USA) and using a reagent of pyridine- d ₅ (Merck, Germany) Respectively.

Through the above analysis, it was determined that the compounds 1 to 4 were the compounds represented by the following formulas 2 to 5, respectively.

Compound 1

(2)

1) Properties: pale yellow wax

2) IR (CaF ₂ , cm ^-1 ): 3,386, 2,932, 1,732, 1,610

3) positive FAB / MS m / z: 487 [M + H] +, C 25 H 43 O 9

4) [?] _D : -2.22 (c = 0.35, MeOH)

5) 1 H-NMR: 5.37-5.46 (6H, m, overlapped, H-7 ", 8 ", 10 ", 11 &

(3H, br s, overlapped, H-1,4 '), 4.40-4.44 (2H, , overlapped, H-2, 2 '), 4.36 (2H, overlapped, H-6'a, H-6'b), 4.31 Overlapped, H-3b), 4.02 (1H, br dd, J = 6.4, 6.0 Hz, H-5 '), 2.87 (4H, br s, overlapped, H-9 '', 12 ''), 2.27 (2H, t, J = 7.6 Hz, H-2 ' 3 ''), 1.26 (4H, br s, H-4 '', 5 ''), 0.88 (3H, t, J = 7.6 Hz, H-16 ''); (C-1 ''), 127.5, 128.2, 128.5, 128.6, 130.1, 132.1 (C-7 ", 8 ", 10 ", 11 & (C-1 '), 77.0 (C-5'), 75.2 (C-3 '), 72.5 (C-2'), 72.2 (C-4 '), 69.0 (C-2), 66.5 (C-1), 62.3 (C-6'), 34.2 C-5 ''), 27.2 (C-4 ''), 25.9 (C-12 ''), 25.9 ) And 14.3 (C-16 ").

Compound 2

(3)

1) Properties: pale yellow wax

2) IR (CaF ₂ , cm ^-1 ): 3,364, 2,931, 1,730, 1,585

3) positive FAB / MS m / z: 515 [M + H] ⁺ , C ₂₇ H ₄₇ O ₉

4) [?] _D : + 3.89 (c = 0.38, MeOH)

5) 1H-NMR: 5.39-5.46 (6H, m, overlapped, H-9 '', 10 '', 12 '', 13 '', 15 '', = 4.6 Hz, H-1 '), 4.51 (2H, d, J = 6.0 Hz, H-1), 4.50 (1H, overlapped, H- (1H, overlapped, H-6'a), 4.36 (1H, overlapped, H-6'b), 4.33 (1H, dd, J = 10.0, 5.2 Hz, H- H-3 '), 4.06 (1H, dd, J = 10.0, 3.6 Hz, H-3b), 4.05 (1H, overlapped, H-5'), 2.86-2.89 overlapped, H-11 '', 14 ''), 2.28 (2H, t, J = 7.6 Hz, H-2 ' ), 1.54-1.57 (4H, m, overlapped, H-3 '', 4 ''), 1.04-1.27 (6H, m, overlapped, H-5 ', 6' 3H, t, J = 7.6 Hz, H-18 '); 13 C-NMR (100 MHz, pyridine-d5, dC) 173.5 (C-1 ''), 127.5, 128.0, 128.6, 128.6, 130.5, 132.0 (C- ', 15'', 16), 105.8 (C-1'), 77.0 (C-5 '), 75.2 (C-3'), 72.5 C-2), 69.0 (C-4 '), 66.5 (C-1), 62.3 5 ''), 29.2 (C-6 ''), 29.2 (C-7 ''), 27.4 (C-8 ' 25.1 (C-3 ''), 20.8 (C-17 '') and 14.5 (C-

Compound 3

[Chemical Formula 4]

1) Properties: pale yellow wax

2) IR (CaF ₂ , cm ^-1 ): 3,399, 2,929, 1,737, 1,590

3) positive FAB / MS m / z: 775 [M + H] ⁺ , C ₄₅ H ₇₅ O ₁₀

4) [?] _D : +11.6 (c = 0.34, MeOH)

5) 1 H-NMR: 5.61 (1H, m, H-2), 5.37-5.49 (12H, m, overlapped, H-9 ", 9 ", 10 ", 10 ", 12 & , 12, 13 '', 13 '', 15 '', 15 ''',16'',16'''), 4.75 (1H, d, J = 7.6 Hz, H- (2H, m, overlapped, H-1b, 4 '), 4.37 (3H, m, H-2', 6 '), 4.07 (1H, overlapped, H-3 '), 4.03 (1H, overlapped, H-3), 3.99 (1H, dt, J = 11.6, 6.0 Hz, H- Overlapped, H-2 '', 2 '''), 2.05 (8H, m, overlapped, M, overlapped, H-3 ', 3''), 1.30 (4H, m, overlapped, H 4 '', 4 '''), 1.23 (12H, m, overlapped, H-5'',5'',6'',6'',7'',7' (6H, t, J = 7.6 Hz, H-18 ", 18 "'); (C-1 '', C-1 '''), 127.2 x 2, 127.7 x 2, 128.2 x 2, 128.2 x 2, 130.2 x 2 , 131.7 × 2 (C-9 '', 9 ''',10'',10''', 12 '', 12, 13 '', 13 ''',15'',15''''', 16 '''), 105.2 (C-1'), 76.7 (C-5 '), 74.8 (C-4 '), 67.7 (C-3), 62.9 (C-1), 61.9 (C-4 '', 4 '''), 29.4 × 2 (C-7'',7''' 5 '', 5 '''), 27.4 × 2 (C-8'',8'''), 25.9 × 2 (C-14 ''', 11 '''), 25.1 × 2 (C-3'',3'''), 20.7 × 2 (C-17 ' , 18 ''').

Compound 4

[Chemical Formula 5]

1) Properties: pale yellow wax

2) IR (CaF ₂ , cm ^-1 ): 3,417, 2,927, 1,736, 1,595

3) positive FAB / MS m / z: 779 [M + H] ⁺ , C ₄₅ H ₇₉ O ₁₀

4) [?] _D : + 0.70 (c = 0.40, MeOH)

5) 1 H-NMR: 5.60 (1H, m, H-2), 5.40-5.50 (8H, m, overlapped, H-9 ", 9 ", 10 ", 10 ", 12 & J = 12.0, 3.2 Hz, H-1a), 4.46 (1H, d, J = 7.6 Hz, 4.8 (3H, overlapped, H-2 ', 6'), 4.31 (1H, dd, J = 10.8, 5.2 Hz, H- , dd, J = 9.6, 3.2 Hz, H-3 '), 4.05 (1H, dd, J = 10.8, 5.6 Hz, H-3b), 4.00 M, overlapped, H, 11 '', 11 ''), 2.30 (4H, t, J = 7.2 Hz, H-2 ' M, overlapped, H-3 ', 3''), 1.62 (4H, m, 4 '', 4 '', 5 '', 5 '', 6 '', 6 ''',7'',7'',15'',15'',16'', 16 '', 17 '', 17 '''), 0.92 (3H, t, J = 7.6, H-18''), 0.84 (3H, t, J = 6.8, H-18''); (C-1 '', C-1 '''), 128.4 x 2, 128.7 x 2, 130.4 x 2, 130.5 x 2 (C-9 '', 9 ''',10'',10''', 12 '', 12, 13 '', 13 ''), 105.7 (C-1 ' (C-2 '), 72.1 (C-2), 70.1 (C-4'), 68.1 (C-3), 63.3 (C-2 '''), 34.2 (C-2''), 31.7 × 2 (C-16'',16'''), 29.9 × 2 29.9 × 2 (C-8 '', 8 '''), 29.6 × 2 (C-4' , 29.3 × 2 (C-7 '', 7 '''), 29.3 × 2 (C-6'',6''' (C-11 '', 11 '''), 25.2 × 2 (C-3'',3'''), 22.8 × 2 (C-18 '', 18 ''').

Cytotoxicity measurement of compounds

To determine the cytotoxicity of the compound obtained in Example 1, cell viability was measured.

First, RAW264.7 cells (Korean Cell Line Bank, Korea) contained 10% FBS (fetal bovine serum), 2 mM glutamate, 100 units / ml penicillin and 100 mg / ml streptomycin (Dulbecco's modified Eagle's medium; WelGENE Inc., Korea) in 5% CO ₂ .

Each of the four kinds of compounds obtained in Example 1 was added to DMEM medium to which no FBS was added to prepare a sample solution. The broth roll A (compound 1) and the broth roll B (compound 2) , 5, 10, 20, 50, and 100 μM, respectively, and the raindrop roll C (compound 3) and the ripening roll (compound 4) were prepared to be 0.5, 1, 2, 5, 10 and 20 μM, respectively.

The RAW 264.7 cells were then seeded in 96-well plates at a density of 1 × 10 ⁴ cells / well and cultured in the same medium for 12 to 18 hours . After that, the medium was removed and the sample solutions for the four kinds of compounds prepared above were each treated, followed by culturing for 20 hours.

10 μl of 10 μg / ml MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (Sigma-Aldrich, USA) reagent was added to each well and left for 4 hours The supernatant was then removed to obtain a precipitate (not treated in blank wells). Then, 100 μl of dimethyl sulfoxide (DMSO) was added to the precipitate, and the formazan formed in the precipitate was dissolved to prepare a solution. After 30 to 60 minutes of the DMSO addition, the absorbance of the solution was measured at 550 nm.

The survival rate of the cells was measured by the ratio of survival cells in the group treated with the invading roll A, the invading roll B, the invading roll C and the invading roll D, respectively, and the control group without any treatment, according to the following equation .

[Equation 1]

Cell viability (%) = [(compound treated cell number OD value - blank well OD value) / (control OD value - blank well OD value)] × 100

As a result, as shown in FIG. 1B, no cell toxicity was observed even at a concentration of 100 μM or higher in the invading roll A and the invading roll B, and the invading roll C and the invading roll D were slightly toxic As shown in Table 1 below, it was confirmed that the LD ₅₀ value was 20 μM or more. The LD ₅₀ value is a value representing a lethal dose for 50 percent kill (LD ₅₀ ). It refers to the amount of a substance that kills 50% of a test animal under a certain condition, and is widely used as an indicator of the degree of toxicity Is used.

Confirming the inhibitory effect of nitrogen monoxide generation on the compound

In order to confirm the anti-inflammatory effect of the compound obtained in Example 1, the degree of inhibition of nitrogen monoxide formation was measured, and the amount of NO produced from RAW 264.7 cells was measured as the form of NO ₂ ^- present in the cell culture medium using Griess reagent .

First, cells and samples were prepared in the same manner as in Example 3.

The RAW 264.7 cells were then seeded in 96-well plates at a density of 1 × 10 ⁴ cells / well and cultured in the same medium for 12 to 18 hours After that, the medium was removed and the sample solution and 1 / / ml LPS (lipopolysaccharide) were treated with the four kinds of compounds prepared above, and then cultured for 20 hours.

100 μl of the cell culture supernatant and 100 μl of Griess reagent were mixed and reacted on a 96-well plate for 10 minutes, and the absorbance was measured at 540 nm.

The Griess reagent was prepared by adding 5% (v / v) phosphoric acid and 0.1% (w / v) naphthylethylenediamine-HCl to 1% (w / v) sulfanilamide It is an added reagent.

Nitric oxide formation inhibitory effect of a compound compound
NO production and cell viability IC ₅₀ (mM) LD ₅₀ (mM) 1 (breaking roll A) 63.8 ± 6.4 > 100 2 (breakdown roll B) 59.4 ± 6.8 > 100 3 (breakdown roll C) 7.7 ± 0.6 > 20 4 (breakdown roll D) 8.0 ± 0.9 > 20

As a result, as shown in FIG. 1A and Table 1, both the flounder roll A to the flounder roll D showed inhibitory activity on the formation of nitrogen monoxide (NO), which is one of the inflammation inducers of inflammatory diseases in a concentration-dependent manner. IC ₅₀ values at 50% inhibitory concentrations were confirmed to be 63.8 μM, 59.4 μM, 7.7 μM and 8.0 μM in the ripening roll A, ripening roll B, ripening roll C and ripening roll D, respectively.

In addition, it was confirmed that the IC ₅₀ values of the above compounds are all in a concentration range that does not show cytotoxicity and can be used without toxicity within an effective range.

A variety of inflammatory Parameter  Confirming expression suppression efficacy

In order to confirm that the four kinds of compounds of the present invention inhibit the expression or production of inflammatory mediators other than the inhibition of nitric oxide production, the compounds of the present invention may be used to treat tumor necrosis factor-alpha (TNF-alpha) ), IL-1β (interleukin-1β) and interleukin-6 (IL-6)

Cells and samples were prepared in the same manner as in Example 3, and RAW264.7 cells were divided into 6-well plates at 1 × 10 ⁵ cells / well, After incubation for 18 hours in the same medium as described above, the medium was removed and the sample solution and 1 / / ml LPS (lipopolysaccharide) were treated with each of the four compounds prepared above and then cultured for 24 hours.

The cultured cells were collected and total RNA (total RNA) was obtained using TRIzol Reagent kit (Invitrogen, USA). CDNA was synthesized from 5 쨉 g of total RNA obtained using MMLV reverse transcriptase (Thermo Scientific, USA) with Moloney murine leukemia virus reverse transcriptase, and PCR was performed using the primers shown in Table 2 below.

Primer sequence mRNA sequence (5 '-> 3') SEQ ID NO: IL -1β Forward TTC ACA GAG GAT ACC ACT CC SEQ ID NO: 1 Reverse GAA GCT GTG GCA GCT ACC TAT GTC T SEQ ID NO: 2 IL -6 Forward GAG GAT ACC ACT CCC AAC AG SEQ ID NO: 3 Reverse TTC ACA GAG GAT ACC ACT CC SEQ ID NO: 4 TNF -α Forward ATG AGC ACA GAA AGC ATG ATC SEQ ID NO: 5 Reverse TAC AGG CTT GTC ACT CGA ATT SEQ ID NO: 6 GAPDH
Forward CGA CTT CAA CAG CAA CTC CCA CTC TTC C SEQ ID NO: 7 Reverse TGG GTG GTC CAG GGT TTC TTA CTC CTT SEQ ID NO: 8

Relative mRNA expression levels were calculated using the ΔΔCT method, The mRNA level of each gene was normalized using GAPDH .

As a result, as shown in FIG. 2, it was confirmed that the expression of IL-1β, IL-6 and TNF-α mRNA was decreased in a concentration-dependent manner by the four types of compound treatment of the present invention.

That is, the compound of Formula 1 of the present invention has an anti-inflammatory effect that inhibits the expression or production of various inflammatory mediators as well as having a whitening activity through inhibition of melanin biosynthesis.

Manufacturing example  1: Preparation of pharmaceutical composition

The pharmaceutical composition containing the compound represented by the above formula (1) (hereinafter referred to as "compound") of the present invention is described, but the present invention is not intended to be limited thereto but is specifically described.

Manufacturing example  1-1. Sanje  Produce

Compound 2 g

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

Manufacturing example  1-2. Manufacture of tablets

100 mg of compound

Corn starch 100 mg

100 mg of milk

Magnesium stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

Manufacturing example  1-3. Preparation of capsules

100 mg of compound

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

Manufacturing example  1-4. Manufacture of rings

Compound 1 g

Lactose 1.5 g

Glycerin 1 g

0.5 g of xylitol

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

Manufacturing example  1-5. Manufacture of granules

Compound 150 mg

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 캜 to form granules, which were then filled in a capsule.

Manufacturing example  2: Preparation of cream containing compound

The atopic cream for improving atopy comprising the compound represented by the formula (1) (hereinafter referred to as "compound") prepared in Example 1-3 was prepared by mixing in the following amounts.

Compound 3 ml

4 ml of glycerin monostearate

4.5 ml of cetearyl alcohol

Stearic acid 3 ml

Wax 1.5 ml

Glycerin 3 ml

Squalane 8 ml

Betaine 6 ml

0.5 ml of sodium hypaloronate

Hardened vegetable oil 2 ml

Polysorbate 60 3 ml

Add 100 ml of distilled water

Although the composition ratio of the ingredients suitable for the formulation of the cosmetic composition is mixed and prepared as a preferable preparation example, the compounding ratio may be arbitrarily varied according to the regional or national preference such as the demand level, the demanded country, and the use purpose.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments, It will be obvious. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Method for Seperation of Compound Derived from Ginseng and          Composition for anti-inflammatory Using the same <130> 1041821 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL1 beta forward primer <400> 1 ttcacagagg ataccactcc 20 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> IL-1 beta reverse primer <400> 2 gaagctgtgg cagctaccta tgtct 25 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> IL-6 forward primer <400> 3 gaggatacca ctcccaacag 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> IL-6 reverse primer <400> 4 ttcacagagg ataccactcc 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TNF alpha forward primer <400> 5 atgagcacag aaagcatgat c 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TNF alpha reverse primer <400> 6 tacaggcttg tcactcgaat t 21 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 7 cgacttcaac agcaactccc actcttcc 28 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 8 tgggtggtcc agggtttctt actcctt 27

Claims (13)

Obtaining an extract from the leaf of ginseng and concentrating it under reduced pressure;
Extracting the ginseng extract concentrated under reduced pressure using ethyl acetate (EtOAc) / water (H ₂ O);
Obtaining and dispersing the water layer with butanol (n-buOH);
Concentrating the water layer and the butanol layer under reduced pressure to obtain ethyl acetate, butanol and water fractions; And
Obtaining the ethyl acetate layer; And
Separating and purifying the ethyl acetate layer to obtain a compound represented by the following formula (2);
&Lt; RTI ID = 0.0 &gt; ginsenoside &lt; / RTI &gt;
(2)

delete The method according to claim 1, wherein the ginseng leaf extract is water, C1-C5 Wherein the extract is extracted with at least one solvent selected from the group consisting of alcohol, ethyl acetate, chloroform, butanol, hexane, and mixtures thereof.
delete delete delete 2. The compound according to claim 1, wherein said compound has anti-inflammatory activity,
Inhibiting the generation of nitric oxide (NO); And
(TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) Inhibiting the production of one or more inflammatory cytokines;
Wherein the inflammation is inhibited through at least one mechanism selected from the group consisting of:
delete delete delete delete delete delete

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