KR20220064360A - Composition for preventing or treating inflammatory diseases comprising extracts of Acorus gramineus and Dendropanax morbifera - Google Patents

Abstract

The present invention relates to a composition for preventing or treating inflammatory diseases comprising an Acorus gramineus extract and a Dendropanax morbiferus extract, and more specifically, to a pharmaceutical composition for preventing or treating inflammatory diseases, having an anti-inflammatory effect that increases in response to a mixture of an Acorus gramineus extract and a Dendropanax morbiferus extract. In the present invention, it was confirmed that when the Acorus gramineus extract and the Dendropanax morbiferus extract are mixed and used in combination, the anti-inflammatory effect significantly increased while cytotoxicity was rather decreased. Accordingly, the Acorus gramineus extract and the Dendropanax morbiferus extract of the present invention can be usefully used for an anti-inflammatory cosmetic composition as well as a pharmaceutical composition or health functional food treating and preventing inflammatory diseases.

Description

Composition for preventing or treating inflammatory diseases comprising extracts of Acorus gramineus and Dendropanax morbifera

The present invention relates to a composition for preventing or treating inflammatory diseases comprising a seokchangpo extract and hwangchil extract, and more particularly, a pharmaceutical composition for preventing or treating inflammatory diseases in which anti-inflammatory efficacy is increased according to a mixture of seokchangpo extract and hwangchil extract is about

Inflammation is a response of living tissues to damage, and the inflammatory response not only plays a key role in the defense and healing of the body that mobilizes the immune system, but is also a very important process involved in the pathology of many diseases. There are countless causes of inflammation, but biological causes such as bacteria, fungi, and viruses are one of them. In addition, during the inflammatory reaction, immune cells, macrophages, interleukin (interleukin-1β, IL-1β) or tumor necrosis factor (tumor necrosis factor-α; TNF-α), such as cytokines (cytokine) or nitric oxide It plays an important role in the reaction process by producing other inflammatory mediators such as (nitric oxide, NO) and prostaglandin (PG). Production of these mediators by macrophages is found in many inflammatory tissues.

For example, when a living body is exposed to an external immune stimulant such as lipopolysaccharide (LPS), which is a bacterial endotoxin, immune cells are activated through a biosignal transduction pathway, and at the same time, inflammation-inducing factors caused by these cells Their expression increases, and as the first step in this process, the mRNA expression of the corresponding factor increases. In addition, increased IL-1β, IL-6, cytokines such as TNF-α, inflammatory mediators and bioenzymes such as iNOS, COX-2, etc. inflammation (inflammation), pain control (pain control), apoptosis (apoptosis) ), tumorigenesis, and autoimmune response, such as pharmacological or physiological biological responses, play an important role.

Overproduction of inflammatory mediators is easily found in many inflammatory diseases such as rheumatoid arthritis, atherosclerosis, chronic hepatitis, pulmonary fibrosis, etc., and is a major factor in the pathology of these diseases. Therefore, the method of inhibiting gene expression for these inflammatory mediators can be a therapeutic principle and method capable of preventing or suppressing various inflammatory diseases.

Inflammatory diseases are reactions in the human body including infection, trauma, and immunological reactions, and there are acute inflammatory symptoms such as fever, flushing, edema, and pain. , changes in cells such as generation of degradative enzymes, transient reactive oxygen species, and sensitized lmphocytes occur. In chronic inflammatory diseases, cell activation and apoptosis occur by infiltration of white blood cells.

On the other hand, hwangchil tree ( Dendropanax morbifera ) is a plant belonging to the family Araliaceae. It is an evergreen broad-leaved arboreous tree that grows only in the southern coastal areas such as Haenam and Wando in Korea and Jeju Island in the world. When the bark is damaged, a yellow resinous liquid is produced, which is called Hwangchil. Hwangchil wood is known to be effective in removing burns and treating eye diseases and burns. Hwangchil tree extract having anticancer activity is described in Korean Patent Publication No. 2000-0004499 as a related technology related to the conventional hwangchil tree, and a hwangchil extract having hepatocellular protective effect, a hwangchil fraction, and a pharmaceutical composition containing them are described. However, it was confirmed that the high concentration of Hwangchil extract showed cytotoxicity.

Acorus gramineus has long been known as a traditional drug used in Korea, Japan, China and other Asian countries to treat various diseases such as digestive problems, depression, epilepsy and central nervous system diseases (Koo, BS, et al . , Biological & Pharmaceutical Bulletin, 26:978-982, 2000). From a pharmacological point of view, it has been reported that seokchangpo has antifungal, antibacterial, insecticidal and neuroprotective properties, and protects against glutamate-induced cytotoxicity when treated to cultured rat neurons. In addition, it has been reported to be used in the prevention and treatment of Alzheimer's disease, protecting against weakened ischemia caused by neuronal death and hyperactivity induced by inhibited methamphetamine (Lee, HS, et al. , Bioresource Technology , 98). :1324-1328, 2007; Cho, J, et al. , Journal of Ethnopharmacology , 73:31-37, 2000; Kwon, YS, et al. , Biological and Pharmaceutical Bulletin , 27:906-909, 2004).

Therefore, in the present invention, as a result of diligent efforts to reduce the toxicity of the hwangchil extract and increase the anti-inflammatory effect, the cytotoxicity is reduced when using a mixed use of the seokchangpo extract and the hwangchil extract, while showing a synergistic effect on the anti-inflammatory effect, The present invention was completed.

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising a seokchangpo extract and hwangchil extract as active ingredients.

Another object of the present invention is to provide a functional health food composition for the prevention or improvement of inflammatory diseases comprising the extract of seokchangpo and hwangchil extract as active ingredients.

Another object of the present invention is to provide an anti-inflammatory cosmetic composition comprising a seokchangpo extract and hwangchil extract as active ingredients.

In order to solve the above problem,

The present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising the extract of seokchangpo and hwangchil extract as active ingredients.

In a preferred embodiment of the present invention, the seokchangpo extract and hwangchil extract are extracted with one or more solvents selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, ethyl acetate, methylene chloride, and mixed solvents thereof. can be

In another preferred embodiment of the present invention, the seokchangpo extract and hwangchil extract may be extracted with water.

In another preferred embodiment of the present invention, the seokchangpo extract is extracted from the rhizome of seokchangpo, and the hwangchil extract may be extracted from the leaves, branches or bark of hwangchil.

In another preferred embodiment of the present invention, the seokchangpo extract and hwangchil extract are 9: 1 (w/w) to 8: 2 (w/w) ratio, preferably 7: 1 (w/w) can be mixed in proportions.

In another preferred embodiment of the present invention, the seokchangpo extract and hwangchil extract produce inflammatory mediators including NO, iNOS and COX-2 and TNF-α, IL-1β and IL-6 It is possible to inhibit the production of inflammatory cytokines, including

In another preferred embodiment of the present invention, the inflammatory disease is dermatitis, allergy, atopy, asthma, conjunctivitis, cataract, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, sepsis, gastric ulcer, gastritis, Crohn's disease, hemorrhoids , ankylosing spondylitis, lupus, fibromyalgia, psoriasis, arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, tendinitis, myositis, hepatitis, cystitis, nephritis, multiple sclerosis, diabetes, scleroderma, gout, neurodegenerative disease, neuroinflammatory disease , silicosis, atherosclerosis, and may be any one or more selected from ischemia.

In another preferred embodiment of the present invention, the inflammatory disease is multiple sclerosis, neuroblastoma, stroke, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, Creutzfeldt-Jakob disease, post-traumatic stress disorder, depression, learning disability, It may be a neuroinflammatory disease selected from the group consisting of cognitive impairment, memory impairment, schizophrenia, and amyotrophic lateral sclerosis.

In addition, the present invention provides a health functional food composition for the prevention or improvement of inflammatory diseases comprising the extract of seokchangpo and hwangchil extract as active ingredients.

In addition, the present invention provides an anti-inflammatory cosmetic composition comprising a seokchangpo extract and hwangchil extract as active ingredients.

In addition, the present invention provides a composition for enhancing cognitive function comprising a seokchangpo extract and hwangchil extract as active ingredients.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cognitive impairment diseases, and a health functional food composition for improving cognitive disorders or memory, comprising the extract of seokchangpo and hwangchil extract as active ingredients.

In the present invention, it was confirmed that the anti-inflammatory efficacy was significantly increased, while the cytotoxicity was rather decreased, when using the mixed use of the extracts of seokchangpo extract and hwangchil extract. It can be usefully used not only in functional foods but also in anti-inflammatory cosmetic compositions.

1 is data confirming the anti-inflammatory effect and cytotoxicity when treated with Seokchangpo extract and Hwangchil extract alone (A: NO generation according to Hwangchil extract treatment, B: Cell viability according to Hwangchil extract treatment, C: NO according to Seokchangpo extract treatment Occurrence, D: cell viability according to treatment with seokchangpo extract).
Figure 2 is data confirming the anti-inflammatory effect and cytotoxicity of the mixed treatment of seokchangpo extract and hwangchil extract (A: NO generation, B: cell viability).
Figure 3 is data confirming the mRNA expression level of inflammatory mediators including iNOS and COX-2 during the mixed treatment of Seokchangpo extract and Hwangchil extract (A: electrophoretic band according to PCR, B: iNOS versus GAPDH mRNA expression level, C: COX-2 mRNA expression level compared to GAPDH).
Figure 4 is data confirming the mRNA expression level of inflammatory cytokines including TNF-α, IL-1β and IL-6 when the Seokchangpo extract and Hwangchil extract mixed treatment (A: electrophoretic band according to PCR, B: compared to GAPDH TNF-α mRNA expression level, C: IL-1β mRNA expression level compared to GAPDH, D: IL-6 mRNA expression level compared to GAPDH).
Figure 5 is data confirming the protein expression level of inflammatory mediators including iNOS and COX-2 during the mixed treatment of Seokchangpo extract and Hwangchil extract (A: Western blot band, B: iNOS protein compared to β-actin Expression level, C: expression level of COX-2 protein compared to β-actin).

As described above, there is a problem in that a high concentration of hwangchil extract shows cytotoxicity. Accordingly, in the present invention, as a result of diligent efforts to select a substance having excellent anti-inflammatory effect without cytotoxicity to reduce the toxicity of the hwangchil extract and increase the anti-inflammatory effect, the cytotoxicity is reduced when using a mixture of seokchangpo extract and hwangchil extract, whereas It was confirmed that there was a synergistic effect on the inflammatory effect.

Accordingly, the present invention relates to a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising the extract of seokchangpo and hwangchil extract as active ingredients.

In the present invention, the seokchangpo extract and hwangchil extract may be extracted with one or more solvents selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, ethyl acetate, methylene chloride, and mixed solvents thereof. In addition, as the extraction method, methods such as room temperature extraction method, hot water extraction method, cold extraction method, reflux extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method, compression method, etc. can be used. The seokchangpo extract and hwangchil extract of the present invention may be preferably characterized in that they are respectively extracted using a hot water extraction method.

In the present invention, the seokchangpo extract is extracted from the rhizome of seokchangpo, and the hwangchil extract is characterized in that it is extracted from the leaves, branches or bark of hwangchil.

In the present invention, the seokchangpo extract and hwangchil extract are 9: 1 (w / w) to 8: 2 (w / w) ratio, preferably 7: 1 (w / w) (or, 8.75: 1.25 ( w/w))).

When the seokchangpo extract and hwangchil extract are expressed in weight ratio, the seokchangpo extract per 100 parts by weight may be included in 85 to 90 parts by weight, preferably 87.5 parts by weight, and the hwangchil extract may be included in 10 to 15 parts by weight, preferably 12.5 parts by weight. .

In the present invention, the seokchangpo extract and hwangchil extract may be characterized in that it inhibits the production of inflammatory mediators including NO, iNOS and COX-2. In addition, the seokchangpo extract and hwangchil extract may be characterized in that it inhibits the production of inflammatory cytokines including IL-1β, IL-6 and TNF-α.

In a specific embodiment of the present invention, Hwangchil hot water extract and Seokchangpo hot water extract were extracted and then cytotoxic and anti-inflammatory activity for each extract was confirmed. As a result, as shown in FIG. 1, it was confirmed that Hwangchil hot water extract inhibited NO generation in a concentration-dependent manner, but showed cytotoxicity at high concentrations. On the other hand, it was confirmed that the seokchangpo hot water extract had no cytotoxicity, and although it had lower anti-inflammatory activity than the hwangchil extract, it was confirmed that it suppressed NO generation in a concentration-dependent manner.

In another specific embodiment of the present invention, when the Seokchangpo hot water extract and Hwangchil hot water extract are mixed, to check whether a synergy effect on the anti-inflammatory effect is shown, the IC ₅₀ concentration value for the NO generation inhibitory effect The two extracts were combined as a reference. The IC ₅₀ value of the Hwangchil hot water extract was 100 μg/ml, and the IC ₅₀ value of the Seokchangpo extract was confirmed to be 700 μg/ml, so the Seokchangpo extract and the hwangchil extract were formulated in a 7:1 (w/w) ratio for reference. As shown in FIG. 2 , it was confirmed that when the seokchangpo extract and hwangchil extract were formulated in a ratio of 7: 1 (w/w), high anti-inflammatory efficacy without cytotoxicity was confirmed. In addition, when the total amount of Seokchangpo hot water extract and Hwangchil hot water extract was 800 μg, the inhibition rate of NO generation was the highest, and it was confirmed that no cytotoxicity was observed.

Furthermore, as a result of measurement using Colby's formula to further verify the synergistic effect of the mixing of Seokchangpo extract and Hwangchil extract, compared to the theoretical value calculated by Colby's formula, Seokchangpo extract and Hwangchil extract 7 : 1 ( w/w) ratio was found to be higher (measured value), which means that when Seokchangpo extract and Hwangchil extract are mixed, anti-inflammatory activity is increased due to the synergistic effect of mixing the two substances.

* In another specific embodiment of the present invention, when Seokchangpo extract and Hwangchil extract were mixed, mRNA and protein expression levels of inflammatory mediators including iNOS and COX-2 were confirmed. As a result, as shown in FIGS. 3 and 5 , it was confirmed that iNOS and COX-2 mRNA and protein expression, which are inflammatory mediators, decreased by the mixed extract of Seokchangpo and Hwangchil. In addition, it was confirmed that the iNOS and COX-2 protein expression was effectively inhibited when the Seokchangpo extract and the hwangchil extract were mixed rather than the Seokchangpo extract and the hwangchil extract alone treatment group.

In addition, when the Seokchangpo extract and Hwangchil extract were mixed, the mRNA expression level of inflammatory cytokines including TNF-α, IL-1β and IL-6 was confirmed, as shown in FIG. 4 , TNF-α, IL-1β And IL-6 production was decreased, and in particular, in the case of TNF-α and IL-1β, it was confirmed that the production significantly decreased in the group treated with the mixed extract of Seokchangpo and Hwangchil compared to the single treatment group.

That is, in the present invention, since it was confirmed that the synergistic effect on the anti-inflammatory effect and the cytotoxicity was reduced when the seokchangpo extract and the hwangchil extract were mixed, it was confirmed that it can be utilized for the treatment and prevention of inflammatory diseases.

In the present invention, the inflammatory disease is dermatitis, allergy, atopic dermatitis, asthma, conjunctivitis, cataract, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, sepsis, gastric ulcer, gastritis, Crohn's disease, hemorrhoids, ankylosing spondylitis, lupus, fibromyalgia , psoriasis, arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, tendinitis, myositis, hepatitis, cystitis, nephritis, multiple sclerosis, diabetes, scleroderma, gout, neurodegenerative disease, silicosis, atherosclerosis and ischemia. There may be more than one.

In addition, the inflammatory disease is multiple sclerosis, neuroblastoma, stroke, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, Creutzfeldt-Jakob disease, post-traumatic stress disorder, depression, learning disability, cognitive impairment, memory impairment, It may be a neuroinflammatory disease selected from the group consisting of schizophrenia, and amyotrophic lateral sclerosis.

Seokchangpo and hwangchil mixed extract contained in 100 parts by weight of the pharmaceutical composition of the present invention may be included in 0.01 parts by weight to 50 parts by weight, preferably 0.01 parts by weight to 25 parts by weight, but is not limited thereto, and cytotoxicity by hwangchil extract does not show, and may be included within the range showing high anti-inflammatory efficacy.

The pharmaceutical composition of the present invention may be formulated in various forms according to conventional methods and used. For example, it may be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and may be formulated in the form of external preparations, suppositories, and sterile injection solutions. In addition, it may be used in the form of external preparations for skin, such as liquids, ointments, creams, lotions, sprays, patches, gels, or aerosols.

It may further include a pharmaceutically acceptable carrier, excipient and diluent depending on each formulation. In addition, according to a conventional method, it can be formulated and used in the form of external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injection solutions, preferably creams, gels, patches, sprays, It may have an ointment, oral preparation, lotion, liniment, pasta, or cataplasma formulation. For example, in the case of an external skin preparation used locally on the site, conventional additives, for example, a preservative, a solvent to aid drug penetration, and an emollient in the case of ointments and creams, such as ethanol or oleyl alcohol, may be included. It may contain conventional carriers.

The carrier, excipient and diluent include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, mineral oil, and the like. When formulating or formulating the pharmaceutical composition, it is usually prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the composition, for example, starch, calcium carbonate, sucrose , lactose, gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

As used herein, the term “administration” refers to providing the pharmaceutical composition of the present invention to a subject by any suitable method. The pharmaceutical composition of the present invention provides an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human as thought by a researcher, veterinarian, doctor or other clinician, that is, alleviation of symptoms of a disease or disorder to be treated. It can be administered in a therapeutically effective amount, which is an amount that induces It is apparent to those skilled in the art that the therapeutically effective dosage and frequency of administration for the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of active ingredients and other ingredients contained in the composition, the type of formulation, the age, weight, and general health of the patient. , sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and various factors including concurrently used drugs. The pharmaceutical composition of the present invention may be administered to an individual by various routes. For example, intravenous, intraperitoneal, intramuscular, intraarterial, buccal, intracardiac, intramedullary, intrathecal, transdermal, enteral, subcutaneous, sublingual or topical administration may be administered, but not limited thereto. The pharmaceutical composition of the present invention may be administered in an amount of 1 to 10,000 mg/kg/day, may be administered once a day, or may be administered in divided doses.

In addition, the present invention relates to a health functional food composition for the prevention or improvement of inflammatory diseases comprising the extract of seokchangpo and hwangchil extract as active ingredients.

In the present invention, the seokchangpo extract and hwangchil extract may be mixed in a ratio of 9: 1 (w/w) to 8: 2 (w/w), preferably 7: 1 (w/w) ratio, Specific details are the same as the pharmaceutical composition for preventing or treating the inflammatory disease.

The seokchangpo extract and hwangchil extract of the present invention may be used as health functional foods, food additives or dietary supplements. When the extract of the present invention is used as a food additive, it may be appropriately used according to a conventional method, such as adding the extract as it is or mixing it with other foods or food ingredients.

In addition, the mixing amount of the seokchangpo extract and hwangchil extract may be suitably changed according to the purpose of use (prevention, health or therapeutic treatment). As a specific example, when preparing food or beverage, the extract of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, when consumed for a long period of time for health and hygiene or health control, it may be added in an amount less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range. there is.

There is no particular limitation on the type of food, but examples of food to which the extract of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and ice cream. There are dairy products, various soups, beverages, teas, drinks, alcoholic beverages, vitamin complexes, etc., and includes all health foods in the ordinary sense.

When the food composition of the present invention is prepared as a beverage, it may contain additional ingredients such as various flavoring agents or natural carbohydrates like a conventional beverage. Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; natural sweeteners such as dextrin and cyclodextrin; Synthetic sweeteners such as saccharin and aspartame may be used. The natural carbohydrate is contained in an amount of 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight, based on the total weight of the food composition of the present invention.

The food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid It may include a carbonation agent used in beverages, and may include, but is not limited to, natural fruit juice, fruit juice for the production of fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The additive ratio is not particularly limited, but is preferably included in the range of 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

In addition, the present invention relates to an anti-inflammatory cosmetic composition comprising a seokchangpo extract and hwangchil extract as active ingredients.

The cosmetic composition of the present invention may further include conventional adjuvants and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments, fragrances, and the like, which are commonly used in cosmetic compositions in addition to the above active ingredients. For example, the cosmetic composition may further include auxiliary components such as glycerin, butylene glycol, polyoxyethylene hydrogenated castor oil, tocopheryl acetate, citric acid, panthenol, squalane, sodium citrate, allantoin, etc. .

Since the cosmetic composition of the present invention is basically applied to the skin, it can be prepared in any formulation conventionally prepared with reference to a cosmetic composition in the art. For example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays, etc. can be formulated, for example, The present invention is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a mask pack, a spray, or a powder.

Furthermore, it relates to a composition for enhancing cognitive function comprising the extract of seokchangpo and hwangchil extract of the present invention as an active ingredient. The cognitive function is characterized in that any one or more consisting of learning ability, memory ability and concentration.

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

These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Manufacture of seokchangpo extract and hwangchil extract

1-1: Preparation of seokchangpo extract

The rhizome of Acorus gramineus (AG) was purchased from Jeju Sanyacho Co (Jeju, Korea). After adding distilled water to 150 g of dried Seokchangpo rhizomes and boiling at 100° C. for 4 hours to extract the extract, the extract was freeze-dried and stored in a refrigerator at 4° C. (yield 152 g). The lyophilized sample was resuspended in PBS before use and then filtered with a 0.22 μM filter.

1-2: Preparation of Hwangchil extract

Hwangchil ( Dendropanax morbiferus ) was prepared by drying the leaves by maintaining the temperature of 50 ℃ ~ 60 ℃ for 3-5 days. After adding 5 liters of water per 1 kg of dried hwangchil leaves, the extract was extracted by boiling at 90° C. for 8 hours. The hot water extraction process was performed twice, and the extracted Hwangchil leaf hot water extract was vacuum-concentrated at 60° C. to 70° C., and then lyophilized and stored.

Confirmation of anti-inflammatory effect of seokchangpo extract and hwangchil extract

2-1: Confirmation of cytotoxicity

In the present invention, the degree of cytotoxicity was measured for each of the hot water extract of Seokchangpo and the hot water extract of Hwangchil obtained in Example 1. BV-2 cell viability was measured using a known method (Kim, JJ, et al. , Journal of Ethnopharmacology , 140:213-221, 2012).

First, a murine microglia cell line BV-2 was used for the experiment, and the BV-2 cells were inactivated by heat with 10% FBS and antibiotics (penicillin 100 U/ml, streptomycin 100 μg/ ml) was prepared by culturing using a DMEM medium containing (37.0° C., 5% CO ₂ and 95% air).

Then, BV-2 cells were plated in a 96-well plate at 1×10 ⁴ cells/well, and then samples with different concentrations were treated. After 1 hour, treatment with LPS (200 ng/ml), a neuroinflammatory factor, and then stimulating BV-2 cells for 24 hours. After treatment, the medium was discarded and 100 μl DMEM containing MTT (500 μg/ml) The medium was added to each well and incubated at 37°C for 4 hours. After incubation, the medium was discarded and DMSO was added to each well to dissolve formazan. The optical density (OD) was measured at 552 nm and compared with the control.

Cytotoxicity by concentration of Hwangchil and Seokchangpo Hwangchil hot water extract Seokchangpo Hot Water Extract Added amount (μg/ml) LPS (200ng/ml) Cell viability (%) Added amount (μg/ml) LPS (200ng/ml) Cell viability (%) - - 100% - - 100% - + 93.51% - + 93.25% 31.25 + 91.72% 31.25 + 91.51% 62.5 + 96.45% 62.5 + 93.38% 125 + 102.58% 125 + 92.31% 250 + 109.56% 250 + 93.37% 500 + 96.71 500 + 91.02 750 + 85.29 750 + 91.91 1000 + 71.84 1000 + 92.57

As a result, as shown in FIG. 1, it was confirmed that the high concentration of Hwangchil hot water extract showed cytotoxicity, whereas the Seokchangpo extract had little cytotoxicity.

2-2: NO production measurement

In the present invention, in order to confirm the anti-inflammatory efficacy of each of the seokchangpo hot water extract and hwangchil hot water extract obtained in Example 1, the nitrite (NO) release inhibitory effect was measured, and the nitrite concentration was measured by a known method (Kim). , JJ, et al. , Journal of Ethnopharmacology , 140:213-221, 2012).

Specifically, BV-2 cells were plated in a 96-well plate at 1×10 ⁴ cells/well, and then, Seokchangpo hot water extract and Hwangchil hot water extract were treated by concentration to analyze the anti-inflammatory effect. After 1 hour, treatment with LPS (200ng/ml), a neuroinflammation factor, and then stimulated BV-2 cells for 24 hours, analyzed whether nitrite (NO) produced at this time had a concentration-dependent inhibitory effect.

For the measurement of nitrite (NO), a NO assay kit (Abcam) using a Griess reagent was used, and the measurement method was carried out according to the instructions for use of the kit. After mixing 100 μl of the cell culture solution and the grease reagent, the reaction was performed for 10 minutes, and the absorbance was observed at 540 nm, and the concentration was finally confirmed using a standard curve.

Effect of inhibiting NO generation by concentration of Hwangchil and Seokchangpo Hwangchil hot water extract Seokchangpo Hot Water Extract Added amount (μg/ml) LPS (200ng/ml) NO generation (μM) Added amount (μg/ml) LPS (200ng/ml) NO generation (μM) - - 3.74 - - 3.92 - + 24.58 - + 25.00 31.25 + 18.72 31.25 + 23.13 62.5 + 14.65 62.5 + 22.87 125 + 10.07 125 + 21.72 250 + 6.90 250 + 18.43 500 + 4.06 500 + 14.81 750 + 3.72 750 + 11.54 1000 + 3.72 1000 + 9.09

As a result, as shown in FIG. 1 , it was confirmed that both Hwangchil hot water extract and Seokchangpo hot water extract inhibit NO generation in a concentration-dependent manner.

Confirmation of synergistic effect due to the mixed use of seokchangpo extract and hwangchil extract

3-1: Confirmation of cytotoxicity

In the present invention, when Hwangchil hot water extract and Seokchangpo hot water extract were mixed, the degree of cytotoxicity was performed in the same manner as in Example 2-1.

The two extracts were blended based on the IC ₅₀ concentration value for the NO generation inhibitory effect of Seokchangpo extract and Hwangchil extract. The IC ₅₀ value of the Hwangchil hot water extract was 100 μg/ml, and the IC ₅₀ value of the Seokchangpo extract was 700 μg/ml, and the concentrations were as shown in Table 3 below.

Cytotoxicity following mixed administration of Seokchangpo extract and Hwangchil extract Hwangchil Extract seokchangpo extract LPS (200ng/ml) Cell viability (%) - - - 100% - - + 96.93 100 - + 104.35 - 700 + 93.04 50 350 + 99.19 100 700 + 104.38

As a result, as shown in FIG. 2, when Seokchangpo hot water extract and Hwangchil hot water extract were mixed at a ratio of 7: 1 (w/w), it was confirmed that the cytotoxicity was reduced compared to the single treatment group.

3-2: NO production measurement

In the present invention, when Seokchangpo hot water extract and Hwangchil hot water extract were mixed, the degree of inhibition of NO generation was performed in the same manner as in Example 2-2 to confirm the synergistic effect.

Anti-inflammatory effect according to mixed administration of seokchangpo extract and hwangchil extract Hwangchil Extract seokchangpo extract LPS (200ng/ml) NO generation Predicted value (%) Actual value (μM) Inhibition rate (%) - - - 3.48 - - - + 24.76 0 100 - + 10.50 57.58 - 700 + 11.24 54.58 50 350 + 7.44 69.94 100 700 + 4.60 81.44 80.73

As a result, as shown in FIG. 2 , when Seokchangpo hot water extract and Hwangchil hot water extract were mixed, it was confirmed that NO generation was more effectively suppressed than in the single treatment group. When greater than the simple sum (expected activity) of each anti-inflammatory activity, it is said to be synergistic. The synergistic predictive action for a mixture of Seokchangpo extract and Hwangchil extract can be calculated as follows using the Colby formula of Equation 1 below (S.R, Colby, "Calculating Synergistic and Antagonistic Responses of Herbicide Combinations", Weeds 1967, 15, 20-22).

[Equation 1]

E = α + β-(α × β÷ 100)

α and β are the measured values of anti-inflammatory activity when the extract is treated alone, and E is the predicted value of the predicted anti-inflammatory activity when α and β are mixed. In the present invention, the predicted value according to the mixed use of Hwangchil extract and Seokchangpo extract was measured by substituting the NO generation inhibition rate into the Colby formula. If the actual value is larger than the predicted value, it can be determined that there is a synergistic effect, and in the present invention, the theoretical value calculated by Colby's equation and the actual value were compared to further verify the synergistic effect of the mixing process.

As shown in Table 4, when 700 ㎍ / ㎖ of seokchangpo extract and 100 ㎍ / ㎖ of hwangchil extract were mixed, the predicted value for the NO generation inhibition rate for Colbysik was calculated to be 80.73%, and the actual NO generation inhibition rate was 81.44%. confirmed to be

That is, since it was confirmed that the actual value was larger than the predicted value, in the present invention, the synergistic effect of anti-inflammatory efficacy according to the mixed use of Seokchangpo extract and Hwangchil extract was confirmed.

Changes in expression of inflammatory response mediators and inflammatory cytokines according to the mixed extract of seokchangpo and hwangchil

4-1: Inhibition of inflammatory response mediators and inflammatory cytokine mRNA expression

In the present invention, when the LPS-stimulated BV-2 cells were treated with a mixed extract of seokchangpo and hwangchil, inflammatory mediators including iNOS and COX-2 and IL-1β, IL-6, and TNF-α were It was confirmed whether the mRNA expression of inflammatory cytokines, including

First, BV-2 cells were aliquoted in a 6-well plate so that 5 × 10 ⁵ cells/well per well, and then treated with Seokchangpo hot water extract and Hwangchil hot water extract by concentration, and 1 hour later, LPS, a neuroinflammation inducing factor (200 ng/ml) and fertilized. After 6 hours, total RNA was isolated using TRIzol reagent. After reverse transcription using 2 μg of RNA, cDNA was prepared using Oligo (dT) 18 primer and AccuPower™ RT Premix. PCR was performed with the gene-specific primers and reaction conditions as shown in Table 5 below, and the expression level of each mRNA was expressed as a relative expression level with respect to the control, GAPDH.

PCR primer sequences and reaction conditions Primer sequence (5'-3') order
number Product size (bp) Annealing
Temperature (℃) cycle iNOS Forward GAG GTA CTC AGC GTG CTC CA One 444 58 28 Reverse AGG GAG GAA AGG GAG AGA GG 2 COX-2 Forward TGA GTG GTA GCC AGC AAA GC 3 319 58 28 Reverse CTG CAG TCC AGG TTC ATG G 4 IL-6 Forward GGA GGC TTA ATT ACA CAT GTT 5 435 58 30 Reverse TGA TTT CAA GAT GAA TTG GAT 6 IL-1β Forward CAA GGA GAA CCA AGC AAC GA 7 428 58 30 Reverse TTG GCC GAG GAC TAA GGA GT 8 TNF-α Forward AGG GAG AGT GGT CAG GTT GC 9 392 58 30 Reverse CAG CCT GGT CAC CAA ATC AG 10 GAPDH Forward ACC ACA GTC CAT GCC ATC AC 11 472 58 28 Reverse CCA CCA CCC TGT TGC TGT AG 12

As shown in FIG. 3 , it was confirmed that iNOS and COX-2 mRNA expression, which are mediators of inflammatory responses, decreased by the mixed extract of Seokchangpo and Hwangchil. In addition, as shown in FIG. 4, it was confirmed that the mRNA expression of inflammatory cytokines including IL-1β, IL-6, and TNF-α was decreased by the mixed extract of Seokchangpo and Hwangchil. In particular, in the case of TNF-α and IL-1β, it was confirmed that the production significantly decreased in the group treated with Seokchangpo and Hwangchil mixed extract compared to the single treatment group.

4-2: Confirmation of ability to inhibit expression of inflammatory response mediators

In the present invention, it was confirmed whether the expression of iNOS and COX-2 proteins, which are mediators of inflammatory responses, was inhibited when the LPS-stimulated BV-2 cells were treated with the mixed extract of seokchangpo and hwangchil.

*First, BV-2 cells were aliquoted in a 6-well plate so that 5 × 10 ⁵ cells/well per well, and then treated with Seokchangpo hot water extract and Hwangchil hot water extract by concentration, and 1 hour later, the neuroinflammation factor It was fertilized by treatment with LPS (200 ng/ml).

After 24 hours, the cells were washed twice with ice cold PBS, and then 50 ml of RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, containing fresh protease inhibitor cocktail) or 100 ml was added to obtain a whole cell lysate. A Bio-Rad DC Protein Assay was used to determine protein concentration.

After electrophoresis of 40 μg of the whole cell lysate on a 10% SDS-PAGE gel, the separated protein was transferred to a PVDF membrane (Millipore; USA). To block non-specific binding, the PVDF membrane was reacted with PBS containing 5% skim milk for 1 hour. PVDF membrane was treated with anti-iNOS antibody (1:1000), anti-COX-2 antibody (1:1000), and anti-β-actin antibody (1:2000) and reacted overnight at 4°C, followed by secondary antibody (horseradish peroxidase-conjugated secondary antibody; 1:1000-2000) was treated and reacted for 1 hour.

Then, the antibody-specific band was observed using an ECL detection kit and a Luminescent Image Analyzer (LAS-3000, Fujifilm, Japan). Each protein expression level was expressed as a relative expression level with respect to the control, β-actin.

As a result, as shown in FIG. 5 , it was confirmed that the expression of iNOS and COX-2 proteins, which are inflammatory mediators, decreased by the mixed extract of Seokchangpo and Hwangchil.

In addition, it was confirmed that the iNOS and COX-2 protein expression was effectively suppressed when the Seokchangpo extract and the hwangchil extract alone were mixed with the Seokchangpo extract and the hwangchil extract alone.

<110> GLOCAL Industry-Academic Cooperation Foundation Konkuk University <120> Composition for preventing or treating inflammatory diseases comprising extracts of Acorus gramineus and Dendropanax morbifera <130> 1067838 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> iNOS_F <400> 1 gaggtactca gcgtgctcca 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> iNOS_R <400> 2 agggaggaaa gggagagagg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2_F <400> 3 tgagtggtag ccagcaaagc 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> COX-2_R <400> 4 ctgcagtcca ggttcatgg 19 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-6_F <400> 5 ggaggcttaa ttacacatgt t 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-6_R <400> 6 tgatttcaag atgaattgga t 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-1b_F <400> 7 caaggagaac caagcaacga 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-1b_R <400> 8 ttggccgagg actaaggagt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNFa_F <400> 9 aggggagagtg gtcaggttgc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNFa_R <400> 10 cagcctggtc accaaatcag 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_F <400> 11 accacagtcc atgccatcac 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_R <400> 12 ccaccaccct gttgctgtag 20

Claims (7)

A composition for inhibiting neuroinflammation, comprising, as active ingredients, a seokchangpo extract and hwangchil extract mixed in a ratio of 7: 1 (w/w).
The method of claim 1,
The seokchangpo extract and hwangchil extract is a composition for inhibiting neuroinflammation, characterized in that it is extracted with one or more solvents selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, ethyl acetate, methylene chloride, and mixed solvents thereof .
As a pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases, the pharmaceutical composition for preventing or treating neuro-inflammatory diseases, comprising as active ingredients an extract of seokchangpo and hwangchil extract mixed in a ratio of 7: 1 (w/w),
The neuro-inflammatory disease is Alzheimer's disease, Parkinson's disease, cognitive impairment, memory impairment, and neuro-inflammatory disease, characterized in that the neuro-inflammatory disease selected from the group consisting of neurodegenerative diseases for the prevention or treatment of pharmaceutical composition for the treatment.
The method according to claim 1, wherein the Seokchangpo extract and Hwangchil extract are extracted with one or more solvents selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, ethyl acetate, methylene chloride, and mixed solvents thereof. A pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases.
A health functional food composition for the prevention or improvement of neuroinflammatory diseases, comprising as active ingredients an extract of seokchangpo and hwangchil extract mixed in a ratio of 7: 1 (w/w),
The neuro-inflammatory disease is Alzheimer's disease, Parkinson's disease, cognitive impairment, memory impairment, and a health functional food composition for the prevention or improvement of neuro-inflammatory diseases, characterized in that the neuro-inflammatory disease selected from the group consisting of neurodegenerative diseases.
7. The method of claim 6,
The seokchangpo extract and hwangchil extract are extracted with one or more solvents selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, ethyl acetate, methylene chloride, and mixed solvents thereof Prevention of neuroinflammatory diseases Or a health functional food composition for improvement.
7 : 1 (w / w) ratio of extracts of seokchangpo and hwangchil extract mixed as an active ingredient comprising a cosmetic composition for anti-inflammatory.

Images