KR101770036B1 - Composition comprising Schizandrae Fructus extract asan effective component for preventing and treatingarthritis - Google Patents
Composition comprising Schizandrae Fructus extract asan effective component for preventing and treatingarthritis Download PDFInfo
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- KR101770036B1 KR101770036B1 KR1020150145022A KR20150145022A KR101770036B1 KR 101770036 B1 KR101770036 B1 KR 101770036B1 KR 1020150145022 A KR1020150145022 A KR 1020150145022A KR 20150145022 A KR20150145022 A KR 20150145022A KR 101770036 B1 KR101770036 B1 KR 101770036B1
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Abstract
The present invention relates to a composition for preventing or ameliorating a disease caused by IL-1 beta containing an extract of Omija as an active ingredient, a health functional food composition for preventing or ameliorating a disease caused by IL-1 beta, a quasi-drug composition, Lt; RTI ID = 0.0 > IL-1. ≪ / RTI > The composition according to the present invention is excellent in the inhibition of inflammation reaction and inhibition of substrate degradation and can be used for the prevention, treatment or improvement of inflammatory diseases or metabolic diseases caused by IL-1 beta. As a natural product treatment agent, toxicity and side effects Can provide a safe therapeutic agent.
Description
The present invention relates to a composition for preventing or ameliorating arthritis caused by IL-1 beta comprising an extract of Omija as an active ingredient. More particularly, the present invention relates to a composition for the prevention or amelioration of arthritis caused by IL-1 beta, which can be used for various fields such as functional foods for preventing or improving arthritis, which contains it as an active ingredient.
IL-1 beta (hereinafter referred to as " IL-1 beta ") is known as a kind of cytokine that promotes inflammatory response through various studies (Punzi L. et al., Crit Rev Clin Lab Sci. , 2002, 39 (1): 63-88). Factor nuclear factor-kappa B (NF-κB) induced by IL-1β regulates a variety of inflammatory factors, including heterodimers composed of p65 and p50 subunits, inactivated NF-κB, (IκB-α) is phosphorylated by stimulation of pro-inflammatory cytokines, chemokines, enzymes, etc. in the cytoplasm in association with inhibitor κB-alpha, α is degraded and NF-κB migrates into the nucleus and binds to the promoter of the target gene to secrete a cytokine by inflammation [Jeong et al., 2010].
According to recent reports, it has been shown that a family of mitogen-activated protein kinases (hereinafter referred to as 'MAPK'), extracellular signal regulated kinase, (P38 MAP kinases) and c-Jun N-terminal kinase (hereinafter referred to as "JNK") were induced by cartilage degradation and MMP It is known to play an important role in cytokine regulation [Thalhamer et al., 2008]. Was found to be the level of MAPK phosphorylation in arthritic cartilage model higher than in normal cartilage, by which through the MAPK than signaling pathways NO, increased
Thus, the relationship between the IL-1β-induced signaling pathway and inflammatory and metabolic diseases is already known. Inflammatory diseases are collectively referred to as inflammatory diseases. The inflammatory diseases include acute and chronic inflammatory diseases. Specifically, the inflammatory diseases include edema, dermatitis, allergy, atopy, asthma, conjunctivitis, rhinitis, otitis media, , Sjogren's syndrome (sjogren's syndrome), gastritis, Crohn's disease, colitis, hemorrhoids, gout, sinus spondylitis, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, shoulder periitis, tendinitis, syndrome and multiple sclerosis.
On the other hand, the joint tissue in the human body is a place where two moving bones meet, and has a smooth cartilage structure for absorbing the impact from the joint. Arthritis is characterized by articular cartilage, decreased osteophyte formation, and subchondral bone hardening, which is a chronic disease in which joint inflammation is caused by edema and pain, and joint stiffness. The most common arthritis is osteoarthritis (degenerative arthritis), rheumatoid arthritis, gouty arthritis, ankylosing spondylitis, infectious arthritis, and arthritis. Small arthritis, achilles tendonitis, and the like. Recent treatments for arthritis include hyaluronic acid, glucosamine, and chondroitin, which are drugs and cartilage regeneration / protection agents that inhibit TNF to control pain and prevent complications.
Metabolic disease refers to diseases caused by imbalance such as carbohydrates, lipids, proteins, vitamins, minerals and water, and includes osteoporosis, diabetes, hypertension, hyperlipemia and heart disease. In this process, an imbalance occurs and osteoporosis occurs. At this time, it is known that IL-1β is involved in osteoclast differentiation and can be a therapeutic target in osteoporosis (A Nemetz, Gut 2001; 49: 644-649).
In order to treat IL-1β-mediated diseases, studies on the inhibition of the IL-1β-induced signal transduction system have been actively conducted, and the use of anti-IL-1R antibodies that are receptors of IL-1β 10-1317045). However, in the case of an anti-IL-1R antibody, it may be an immunogenic when used as a therapeutic agent, since it may have an epitope which is a part that can be recognized as an exogenous protein when introduced into an individual. In order to solve the above problems, many studies have been conducted to develop a therapeutic agent for IL-1β-mediated diseases using a small molecule compound that is not a protein not recognized by the immune system.
Although the pharmacological composition has been developed as a natural material, most of the products have been commercialized using the extract, and thus the exact mechanism of pharmacological action has not been clarified. However, since it is easy to take as an oral preparation and has little toxicity and side effects, .
Under these circumstances, the present inventors have made efforts to find a natural product targeting a pathway activated by IL-1 beta. As a result, it has been confirmed that the extract of Omija effectively inhibits the signal transduction system induced by IL-1 beta, Thereby completing the invention of a composition for preventing or ameliorating diseases caused by IL-1? Contained as an active ingredient.
It is an object of the present invention to provide a composition for preventing or ameliorating arthritis caused by IL-1 beta, which is capable of inhibiting inflammatory reaction and substrate degradation in an inflammatory or metabolic disease model by IL-1 beta.
Another object of the present invention is to provide a composition for preventing or ameliorating arthritis caused by IL-1 beta, which can be used in various fields such as a health functional food composition capable of preventing or improving arthritis.
In order to achieve the above object, the composition for preventing or improving arthritis caused by IL-1β according to an embodiment of the present invention comprises an extract of Omija as an active ingredient.
The above-mentioned Omiza extract may be extracted with a solvent selected from the group consisting of water, an alcohol having 1 to 6 carbon atoms, and a mixed solvent thereof.
The arthritis may be osteoarthritis or rheumatoid arthritis.
In the composition for preventing or ameliorating arthritis caused by IL-1β, the above-mentioned Omija may be selected from the group consisting of Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium bifidum bifidobacterium, Bifidobacterium bifidobacterium, Bifidobacterium breve bifidobacterium, and Lactobacillus acidophilus may be inoculated and inoculated in the fermented broth.
The Omija may be an Omija seed.
The composition for preventing or ameliorating arthritis caused by IL-1 [beta] may further comprise Bacillus anthracis extract.
The quasi-drug composition for preventing or ameliorating arthritis caused by IL-1? By IL-1? According to another embodiment of the present invention may include a composition for preventing or ameliorating arthritis caused by IL-1?.
The food composition for preventing or ameliorating arthritis caused by IL-1? By IL-1? According to another embodiment of the present invention may include a composition for preventing or ameliorating arthritis caused by IL-1?.
Hereinafter, the present invention will be described in more detail.
The composition for preventing or ameliorating diseases caused by IL-1? According to an embodiment of the present invention comprises an extract of Omija as an active ingredient.
The scientific name of Schizandra chinensis (Turcz.) Baill. , And it is a deciduous broad-leafed vine tree. Leaves are alternate ovate or obovate, with sharp tip, serrate, and hairs on the back side. Flowers bloom in June to July as reddish white, fruit is used for food, and is wild throughout the country excluding Chungnam and Chungbuk, and is distributed geographically in Asia including Japan, Sakhalin, Manchuria and China. Fruits include Schizandrol, Schizandrin A, B, C, angeloylgomisin O, P, epigomisin, pregomoisin, deoxyschizandrin ) And the like. Omija has long been used as a raw material for herbal medicines because of its medicinal properties such as astringency, nourishment, tonic, herbal medicine, ginger poison, thirst, astringent, Human stability due to ingestion has already been confirmed. Omiza extract stimulates the central nervous system to enhance the tonic and intellectual activities and labor capacity. It affects the metabolism of the carbohydrate and accelerates the digestion of the liver glycogen, thereby increasing the content of glycogen, promoting fatigue recovery, regulating blood pressure, It is used for hypotension, arteriosclerosis, diabetes, hepatitis, etc. It is also used in cosmetics because it has antioxidative effect, skin aging inhibition, antibacterial and whitening effect. In the present invention, omija can be commercially sold, purchased in nature or cultivated.
The term 'IL-1β-mediated disease' means a disease in which IL-1β binds to a receptor of IL-1β, induces the activity of MAPK and NF-κB, and expresses a target gene thereof. The disease caused by IL-1 [beta] is not limited thereto, but may be an inflammatory disease or a metabolic disease. The inflammatory diseases are collectively referred to as osteoarthritis, rheumatoid arthritis, psoriatic arthritis, atopic dermatitis, psoriasis, asthma, systemic lupus erythematosus, and multiple sclerosis , Preferably osteoarthritis or rheumatoid arthritis. The metabolic diseases are collectively referred to as diseases caused by metabolic disorders in vivo, and they may be selected from the group consisting of osteoporosis, atherosclerosis and myocardial infarction, preferably osteoporosis.
IL-1β binds to IL-1R, a receptor for IL-1β, and then regulates substrate degradation-related MMP and inflammation-related iNOS, COX-2 via downstream MAPK and NF-κB. The extract of Omiza of the present invention has an activity of inhibiting the expression of p38 and JNK, which are MAPK family, in signal transduction by IL-1β and inhibits the degradation of IκB-α as an inhibitor of degradation of IκB-α Lt; RTI ID = 0.0 > NF-kB < / RTI > activity (Figures 6 and 7).
In one embodiment of the present invention, the Omiza extract of the present invention inhibits the expression of PGE 2 (prostaglandin E 2 ) synthesized from NO (nitric oxide), COX-2 and arachidonic acid, which are increased by inflammation-inducing iNOS and iNOS , And inhibited the inflammatory reaction. From these results, it can be seen that the extract of Omiza of the present invention can effectively prevent or treat osteoarthritis and rheumatoid arthritis, which are typical inflammatory diseases, and can be effectively used for the treatment of diseases caused by IL-1β such as inflammatory diseases Respectively.
In one embodiment of the present invention, it was confirmed that the extract of Omiza of the present invention effectively inhibited the activity of MMP which degrades the extracellular matrix of the connective tissue and the main component of the basement membrane. MMP is known to be a collagenase secreted by fibroblasts, polymorphonuclear leukocytes, epithelial cells and macrophages, and parathyroid hormone and intracellular toxin, prostaglandins, in association with bone resorption, increase the synthesis of MMP. The present invention provides a pharmaceutical composition suitable for bone metabolism diseases by protecting cartilage by inhibiting collagenase by decreasing the expression of MMPs, which are target genes of MAPK family and MAPK, particularly MMP-1, MMP-3 and MMP-13 (Figs. 3 and 5). From these results, it was confirmed that the present invention extract of Omiza can effectively prevent or treat osteoarthritis, rheumatoid arthritis and osteoporosis, which are representative metabolic diseases, which are inflammatory diseases or metabolic diseases. It may be effective for treatment.
In the present invention, " prevention " means any action that inhibits or delays disease caused by IL-1? By administration of the composition, and " treatment " Or to change or improve the symptoms of the disease.
In the present invention, the Omiza extract may be extracted with a solvent selected from the group consisting of water, an alcohol having 1 to 6 carbon atoms, and a mixed solvent thereof. The term "Omiza extract" means an extract obtained by extracting Omiza. The above-mentioned Omiza extract is prepared by mixing the ground powder with a polar solvent such as an alcohol having a carbon number (C 1 ) to (C 6 ) such as water, ethanol, methanol or the like or a mixed solvent having a mixing ratio of alcohol and water of 1: 0.1 to 1:10 And may be eluted with a mixed solvent having a mixing ratio of ethanol and water of preferably 1: 3 to 5. At this time, the extraction temperature may be an extract extracted at 10 to 100 캜, preferably at room temperature, and for an extraction period of 12 to 4 days, preferably 24 hours. The extract may be a result obtained by concentration under reduced pressure with a vacuum rotary concentrator. However, as long as it is an extract of Omija that can exhibit the therapeutic effect of the disease caused by IL-1? Of the present invention, A concentrate, a dried product obtained by drying the extract, or a controlled preparation or a purified product thereof. In addition, in Omiza, it can be extracted from various organs of natural, hybrid, and variant plants.
Preferably, the ethanol extract for Omija seeds may be used. This is because the inhibitory effect on IL-1β is superior to that of the extracts from other sites. More preferably, the ethanol may be 12 to 22 wt%. The extract according to the above range may be most advantageous for obtaining the active ingredient.
The Schizandra chinensis extract may have been extracted using fermented Schizosaccharomyces rubrum. When the fermented Schizosaccharomyces pombe is used, it has a low cytotoxicity and can be used stably at a relatively high concentration.
Examples of the microorganism used for the fermentation include Lactobacillus salivarius, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus rhamnosus, Lactobacillus plantarum Lactobacillus plantus, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus delbrueckii, Such as Lactobacillus reuteri, Lactobacillus buchneri, Lactobacillus gasseri, Lactobacillus johonsonii, Lactobacillus kefir, and the like, lactic acid bacteria such as lactobacillus kefir, Lactococcus lactis, Lactococcus plantarum, Lactococcus raffinolactis, Enterococcus faecalis, Enterococcus faecium, Streptococcus thermophilus, Streptococcus spp. Bifidobacterium bifidum, Bifidobacterium bifidum, Bifidobacterium bifidum, and Bifidobacterium bifidum, such as Leuconostoc lactis, Leuconostoc mesenteroides and the like, Bifidobacterium species such as Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium pseudolongum, Bifidobacterium themophilum, Bifidobacteria such as Bifidobacterium adolescentis, and the like, Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium bifidum bifidobacterium, Bifidobacterium breve bifidobacterium, and Lactobacillus species such as Lactobacillus casei, Lactobacillus rhamnosus, And Lactobacillus acidophilus. ≪ Desc /
The pharmaceutical compositions of the present invention may comprise a pharmaceutically acceptable carrier. Compositions comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration may include tablet pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like. In addition, the pharmaceutical composition of the present invention may be formulated into tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, And can have any one of the formulations selected.
The Omiza extract of the present invention has been used for edible and medicinal purposes from the past, and there is no particular limitation on the dosage thereof, and there is no particular limitation on the amount of the body extract, body weight, age, sex, health condition, diet, , The severity of the disease, and the like. In general, the Omija extract is preferably administered in an amount of about 10 to 1000 mg per kg of body weight, and more preferably about 50 to 500 mg per kg of body weight. The pharmaceutical composition containing the extract of Omija as an active ingredient of the present invention is prepared considering the effective dose range. The unit dosage formulations thus formulated are classified according to the judgment of the expert who observes or observe the administration of the drug, , Or may be administered several times at regular intervals.
The present invention also provides a quasi-drug composition for preventing or ameliorating a disease caused by IL-1β comprising an extract of Omija as an active ingredient. The production and composition of the above Omiza extract are as described above. More specifically, the extract of Omiza of the present invention can be added to a quasi-drug composition for the purpose of prevention or treatment of diseases caused by IL-1 ?.
In the present specification, the term 'Bok Jo-hui grass' is a deciduous shrub belonging to the buttercup butterfly. The leaf is a three-folded leaf composed of three small leaves. The small leaf is wide egg-shaped, and two of the three are next to each other. The leaves grow larger as they go up to the top, with sharp ends and coarse teeth, with slightly reddish ridged sawtooth on the edge, but are often divided into three shallowly.
In the case of using the above-mentioned Bacillus subtilis paste, it is effective for washing and improving skin wrinkles. However, it is necessary to remove the toxicity through the fermentation process because the bacteriocin has its own toxicity.
Preferably, the baculovirus full extract may be a full-fermented bacillus subtilis extract prepared by a fermentation extraction method. Since the bacillus grass is weakly toxic, it is necessary to remove the toxicity through the fermentation process.
In the present invention, the full fermentation extract of Bacillus thuringiensis is prepared by adding 1 to 3 parts of saccharide of skin and 0.05 to 0.25 part of suntan oil to 100 parts of rice sole, and adding 0.5 to 2.0 parts of skin microbial solution The whole pulverized product can be obtained by immersing the pulverized product in a volume of 10 to 30 parts by volume and then fermenting at room temperature for 3 to 15 days in a room temperature atmosphere.
The microorganism may be selected from the group consisting of Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium bifidum bifidobacterium, Bifidobacterium breve bifidobacterium, and Lactobacillus sp. Lactobacillus acidophilus, and Bacillus acidophilus.
As used in the present invention, 'kangsak' is a perennial plant belonging to the rose family. The height is about 30 to 100 centimeters. The stem grows from the coarse rootstock and has hairs throughout. Leaves are alternate phyllotaxis, 5 ~ 7 small leaves. In June ~ August, a yellow flower blooms in total sprout, and the fruit has thorny hairs and sticks to other things.
In the present invention, the term 'quasi-drug' means a fiber, a rubber product or the like used for the purpose of treating, alleviating, treating or preventing a disease of a human or an animal, a weak action against the human body, Used for the purpose of diagnosing, curing, alleviating, treating or preventing diseases of human or animal, which are not machinery, similar products, products for sterilization, insecticide and similar uses for the prevention of infectious diseases. Machinery, or apparatus, and that is not an apparatus, machine or apparatus of an article used for the purpose of causing pharmacological effects on the structure or function of a person or animal.
When the omija extract of the present invention is used as a quasi-drug additive, the above omija extract may be added as it is or may be used together with other quasi-drugs or quasi-drugs, 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 may be, but is not limited to, disinfectant cleaner, shower foam, gagrin, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment or filter filler.
The present invention also provides a health functional food composition for preventing or ameliorating a disease caused by IL-1 [beta] comprising an extract of Omija as an active ingredient. The production and composition of the above Omiza extract are as described above. More specifically, the Omiza extract of the present invention can be added to a health functional food composition for the purpose of preventing or treating diseases caused by IL-1 ?.
When the Omiza extract of the present invention is used as an additive for health functional food, the Omiza extract may be added as it is or may be used together with other health functional food or health functional food ingredients, and may be appropriately 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.
There is no particular limitation on the kind of the health functional food of the present invention. Examples of the health functional food to which the above extraction mixture can be added include meat products, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen noodles, other noodles, gums, dairy products including ice cream, , A drink, an alcoholic beverage, and a vitamin complex, and may include foods used as food for animals, which may include all health functional foods in the conventional sense. In addition to the above, the health functional food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, , Alcohols, carbonating agents used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks.
The present invention also provides a method for treating a disease caused by IL-1 [beta], comprising the step of administering the composition to a subject suspected of having a disease caused by IL-1 [beta] other than human.
In the present invention, the suspected individual of IL-1β refers to all animals that have developed or are capable of developing an inflammatory disease or metabolic disease. The pharmaceutical composition comprising the extract of Omija of the present invention may be administered to a subject suspected of having a disease caused by IL- , The individual can be treated efficiently. The diseases caused by IL-1 [beta] are as described above.
In the present invention, 'administering' means introducing the pharmaceutical composition of the present invention into a subject suspected of having a disease caused by IL-1β by any appropriate method. The administration route may be any pathway of oral or parenteral routes ≪ / RTI >
The method of treatment of the present invention may include administering a pharmaceutical effective amount of a pharmaceutical composition comprising an extract of Omija as an active ingredient. The appropriate total daily dose may be determined by the treatment within the scope of appropriate medical judgment, and may be administered once or several times. For purposes of the present invention, however, the specific therapeutically effective amount for a particular patient will depend upon the nature and extent of the reaction to be achieved, the particular composition, including whether or not other agents are used, the age, weight, Sex and diet of the patient, the time of administration, the route of administration and the rate of administration of the composition, the duration of the treatment, the drugs used or concurrently used with the specific composition, and similar factors well known in the medical arts.
Inhibition of MAPK family and NF-κB activity by the composition for preventing or ameliorating diseases caused by IL-1β, which comprises the extract of Omija of the present invention as an active ingredient, inhibits iNOS, NO, COX-2, PGE 2 , MMP expression can be inhibited to protect the substrate and inhibit the inflammatory response.
Accordingly, the present invention can be applied to various fields such as health functional foods that can prevent and improve arthritis by using a composition for preventing or ameliorating diseases caused by IL-1 [beta].
FIG. 1 shows the results of a cytotoxicity test according to an embodiment of the present invention.
FIG. 2 shows the results of cytotoxicity test by the treatment with the fermented extract of Omija, according to an embodiment of the present invention.
FIG. 3 shows the results of inhibition of PGE 2 , MMP-1, MMP-3 and MMP-13 production by the treatment with Omija extract according to an embodiment of the present invention.
FIG. 4 shows the results of inhibition of iNOS and COX-2 production by the treatment of Omija extract according to an embodiment of the present invention.
FIG. 5 shows the results of suppressing the production of MMP-1, MMP-3 and MMP-13 by the treatment with Omija extract according to an embodiment of the present invention.
FIG. 6 shows the expression of p65 and IκB-α in nucleus and cytoplasm according to an embodiment of the present invention.
FIG. 7 shows the changes of p-Akt, p-JNK, p-ERK and p-p38 by the treatment with Omija extract according to one embodiment of the present invention.
It is to be understood that both the foregoing general description and the following examples are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. It will be apparent to those skilled in the art that such variations and modifications are intended to fall within the scope of the appended claims.
<Examples>
<Preparation of Omiza Extract>
The omija seeds were washed thoroughly with water, stored at -20 ° C, lyophilized, and homogenized using a grinder. The homogenized Schizochytrium was extracted with 20w% ethanol (RT) for 24 hours (EESF) and filtered. The filtered extract was concentrated in a rotary vacuum concentrator (Buchi Rotavapor R-144, BUCHI Labortechnik, Flawil, Switzerland) The solvent was removed and Omija extract was obtained as the extracted residue. The extract was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich Chemical Co., St. Louis, Mo., USA) at a concentration of 50 mg / ml. The above-mentioned Omiza extract solution was stored at 4 캜 and used in the experiment.
Also, an extract of fermented Omija was prepared by extracting the same as the above-mentioned method for producing Omiza extract, using Omija seeds fermented for 5 days by inoculation with Lactobacillus casei.
<Cell Culture>
SW1353 chondrocytes were purchased from ATCC (American Type Culture Collection, Rockville, Md., USA) and incubated with 100 units / ml penicillin, 100 mg / ml streptomycin, 10% fetal bovine serum bovine serum) was cultured in a 5% CO 2 incubator at 37 ° C in DMEM (Dulbecco's Modified Eagle's Medium).
<Experimental Example>
<Experimental Example 1> Cytotoxicity measurement by treatment with Omiza extract
MTT assay was used to investigate the effect of Omiza extract on SW1353 chondrocyte survival. SW1353 chondrocytes were plated at 3 × 10 5 cells / ml in the wells of the plate and the Omiza extract was treated with the concentrations of 100 μM, 300 μM, 500 μM, 700 μM, 1000 μM / Respectively. Thereafter, the medium of each well was removed, and MTT reagent of 0.5 mg / mL was added thereto, followed by incubation for 2 hours, and then the supernatant was removed. DMSO (Dimethylsulfoxide) was added to the formazan crystals produced in each well and the optical density was measured with a microplater reader at 540 nm.
In addition, the frequency divider SW1353 chondrocyte to 3 × 10 5 cells / ml in the well (well) of the plate, the Schisandra chinensis extract by concentration in the medium after a pre-treatment for 1 hour (100, 300, 500㎍ / ㎖ ), 40ng MTT assay was performed in the same manner as in the case of the IL-1β treated group (experimental group) and the untreated control group (control group).
1A, when the extract of Omija was treated at a concentration of 500 μg / ml or less, the cell survival rate was 100% or more, and even when treated at a concentration of 700 μg / ml, the cell viability was 70% or more, It can be applied. In addition, referring to FIG. 1B, regardless of IL-1? Treatment, it was confirmed that a high cell survival rate of 95% or more was obtained when 0-500 占 퐂 / ml Omiza extract was treated.
In addition, the cytotoxicity of the fermented Omija extract was tested by the same method, and the results are shown in Fig. As a result, it can be confirmed that the fermented Omija extract is not cytotoxic even at a relatively high concentration and can be used without side effects.
<Experimental Example 2> Measurement of NO (nitric oxide) inhibitory activity by treatment with Schizandra chinensis extract
The nitrite assay was used to investigate the effect of Omija extract on NO production in SW1353 chondrocytes. SW1353 chondrocytes were plated in wells of the plate and the Omija extract was pretreated for 1 hour at concentrations of 100, 300 and 500 占 퐂 / ml and then treated with 40ng / ml of IL-1? (Experimental group) and untreated group (control group) were cultured for 24 hours. The NO 2 concentration used as an indicator of NO synthesis was determined using a Griess reagent and the absorbance was measured at 540 nm with a microplater reader.
As a result, the value of NO (μm) was 43 (μg / ml) at 100 μg / ml, 72 (μm) at 300 μg / (μm). Therefore, it was confirmed that the inhibitory activity against the nitrogen oxide was increased in the concentration-dependent manner in the above-mentioned Omiza extract.
In addition, it was confirmed that the extract of the fermented Omija extract had excellent nitrogen oxidation activity from 100 μg / ml to 70 μl, from 300 μg / ml to 61 μl, and from 500 μg / ml to 39 μl. Therefore, the use of Omija fermented extract is advantageous because it is low in cytotoxicity and can be provided without side effects.
≪ Experimental Example 3 > The PGE 2 , MMP inhibitory activity measurement
The inhibitory activity of PGE 2 , MMP-1, MMP-3, and MMP-13 of Omiza extract was measured using SW1353 chondrocytes. SW1353 chondrocytes were plated in wells of a plate, and Omija extract was pretreated for 1 hour at concentrations (100, 300, 500 占 퐂 / ml) and then treated with 40 ng / ml of IL-1? (Experimental group) and the untreated group (control group) were cultured for 24 hours. The amounts of PGE 2 , MMP-1, MMP-3 and MMP-13 in the supernatant were measured by ELISA kit (R & D systems, Minneapolis, MN, USA).
3 A and 3 C, the amount of MMP-1 was significantly increased by IL-1β, and the amount of MMP-1 and MMP-13 was 60% Or more. In addition, as shown in FIG. 3B, the amount of MMP-3 induced by IL-1β was decreased by 20% or more by the treatment with Omiza extract. Therefore, it was confirmed that Omiza extract reduces cartilage tissue by decreasing PGE 2 , MMP-1, MMP-3, and MMP-13 levels and alleviating the inflammatory reaction and preventing degradation of extracellular matrix and basement membrane of connective tissue.
< Experimental Example 4> by the treatment of Omiza extract iNOS , COX-2, MMP mRNA And protein inhibitory activity
The expression of iNOS, COX-2, MMP-1, MMP-3, and MMP-13 in Omija extract was measured using SW1353 chondrocytes. SW1353 chondrocytes were plated in wells of a plate, and Omija extract was pretreated for 1 hour at concentrations (100, 300, 500 占 퐂 / ml) and then treated with 40 ng / ml of IL-1? (Experimental group) and the untreated group (control group) were cultured for 24 hours.
EXPERIMENTAL EXAMPLE 4-1 Identification of mRNA Inhibitory Activity of iNOS, COX-2 and MMP
RNA was isolated from the cultured SW1353 chondrocytes using TRIzol reagent (Invitrogen, CA, USA), and cDNA was synthesized using M-MLV reverse transcriptase (Promega, Madison, WI, USA) Was used for the experiment.
RT-PCR was carried out using the synthesized cDNA and the primer shown in Table 1 below, and the product was electrophoresed on 1% agarose gel and imaged by EtBr staining. The primers of iNOS, COX-2, MMP-1, MMP-3, MMP-13 and GAPDH (used as controls) used in the experiment are shown in Table 1.
From the results of FIG. 4B, it was found that the Omiza extract decreased the mRNA levels of iNOS and COX-2, and that the mRNA levels of iNOS, COX-2, MMP-1 and MMP- 5B, Omija extract also decreased mRNA levels of MMP-1, MMP-3 and MMP-13.
< Experimental Example 4-2> iNOS , COX-2, MMP Identification of protein inhibitory activity
Nuclear and cytoplasmic proteins were extracted from the cultured SW1353 chondrocytes using nuclear extraction reagents (Pierce, Rockford, Ill., USA). The extracted proteins were dissolved in lysis buffer (0.5% Triton, (PH 7.2), 0.1 mM Na 3 VO 4 , 2 mM MgCl 2 , 1 mM EGTA, 1 mM DTT, 2 μg / ml leupeptin, 0.1 mM PMSF and 4 μg / ml aprotinin (aprotinin). Protein concentration was quantitated using a protein assay kit (Bio-Rad protein assay kit, Bio-Rad, Hercules, Calif., USA) and analyzed by SDS-polyacrylamide gel Protein was electrophoresed by loading 40.. After electrophoresis, the proteins of the gel were transferred to NC (nitrocellulose, Schleicher and Schuell, Keene, NH, USA) membranes. The transferred membrane was blocked with 5% nonfat dry milk at room temperature for 1 hour, and the primary antibody was treated overnight at 4 ° C. The membrane was then reacted with HRP-conjugated secondary antibody and developed in an dark room using an ECL kit (Amersham Corp., Arlington Heights, IL, USA).
As a result, it was confirmed from Fig. 4A that the Omiza extract decreased the protein levels of iNOS and COX-2 and that the Omiza extract also decreased the protein levels of MMP-1, MMP-3 and MMP-13.
Therefore, it has been shown that the Omiza extract inhibits the expression of iNOS and COX-2 induced by IL-1 [beta], thus also inhibiting the production of NO regulated by iNOS (Experimental Example 2). It was also confirmed that the extract of the present invention as a natural product has an effect of inhibiting and alleviating the inflammatory reaction by reducing COX-2 required for the synthesis of prostaglandins synthesized from arachidonic acid.
In addition, since the extract of Omija reduces the expression of MMP-1, MMP-3 and MMP-13 induced by IL-1β, it prevents the secretion of collagenase secreted by fibroblasts and macrophages, By inhibiting collagen degradation of the extracellular matrix and basement membrane of cartilage tissue, cartilage tissue can be protected and cartilage degradation can be suppressed.
< Experimental Example 5> by the treatment of Omiza extract NF - κB Confirmation of the movement of p65 into the nucleus
In the same manner as the above protein inhibitory activity assay, 500 μg / ml of Omija extract was pretreated for 1 hour, and 40 ng / ml of IL-1β was treated according to time (for 15, 30 and 60 minutes respectively) The expression of p65 in the nucleus of SW1353 chondrocytes and the expression of IκB-α in the cytoplasm were examined. According to Fig. 6A, the expression of p65 in the nucleus was increased by IL-1? Treatment and the expression of p65 in the nucleus was decreased over time when the extract of Omiza was treated together. In addition, the expression of IκB-α in the cytoplasm was decreased by treatment with IL-1β, but the expression of IκB-α in the cytoplasm was increased when the Omiza extract was treated together. This means that the Omiza extract inhibits the degradation of IκB-α and inhibits the migration of p65 to the nucleus by IL-1β.
< Experimental Example 6> by the treatment of Omiza extract NF - κB Confirmation of the movement of p65 into the nucleus
Immunofluorescence was used to confirm the extent of NF-κB migration into the nucleus when the Omiza extract was treated. SW1353 chondrocytes were plated on a cover glass on a well of a 6-well plate and the medium was pretreated with 500 mu g / ml Omija extract for 1 hour and then treated with 40 ng / ml IL-1 beta (experimental group) (Control group) were cultured for 30 minutes. Cells were washed twice with PBS (phosphate buffered saline) and fixed with 4% paraformaldehyde for 10 min at room temperature. The immobilized cells were then permeabilized with methanol at 20 ° C for 10 minutes, washed twice more with PBS, and then treated with NF-? B p65 (1: 100 dilution) primary antibody overnight at 4 占 폚. Subsequently, FITC-conjugated anti-rabbit IgG (1: 200 dilution) was reacted for 1 hour, washed with PBS, stained with DAPI solution, and imaged using a fluorescence microscope (Carl Zeiss, Jena, Germany) Respectively.
From FIG. 6B, the control group shows that NF-κB was transferred into the nucleus by IL-1β treatment, and when pretreated with Omija extract, NF-κB was hardly visible in the nucleus compared to the control group. It inhibits the degradation of IκB-α by the action of Omiza extract and inhibits the migration of NF-κB into the nucleus, thereby regulating the expression of NF-κB target genes such as inflammatory response-related genes such as NO and PGE 2 and MMP Or suppression of the disease.
< Experimental Example 7> By the treatment of Omiza extract Akt , MAPK In the family Identification of protein inhibitory activity
In the same manner as the method for confirming the protein inhibitory activity, the extracts of 100, 300 and 500 μg / ml of Omija were pretreated for 1 hour, treated with 40 ng / ml of IL-1β for 1 hour, The protein levels of p38, JNK, and ERK were confirmed. As a result, it was confirmed that the phosphorylated p-JNK and p-p38 were reduced in concentration-dependent manner upon treatment with Omiza extract. Thus, Omiza extract may induce a decrease in the signaling pathway mediated by JNK, p38, among IL-1β-mediated signal transduction.
< Experimental Example 8-1> Preparation of complex extract
The fermented Omija seed extract B fermented with the above-mentioned Omiza seed extract A and Lactobacillus casei was prepared.
The hot - water extract (Extract C), Nankia 's hot water extract (Extract D) and hot water extract (Extract E) for mung bean were prepared.
The extracts were then mixed according to the following Table 1 to prepare Examples.
(Unit: parts by weight)
<Experimental Example 8-2> Sensory evaluation on the compound extract
The above complex extracts T1 to T10 were prepared as a beverage, and a sensory evaluation was performed on 20 adult male and female. The sensory evaluation was evaluated by an index of 1 to 10 according to palatability on the basis of flavor and taste, and classified by an average index. The results are shown in Table 2. (The higher the number is, the higher the palatability will be)
(Unit: index)
Referring to the above Table 2, it can be confirmed that the fermented Omiza has a poor flavor and poor palatability as compared with Omiza. However, the flavor of the fermented soybean paste was higher than that of the fermented soybean paste, and the flavor of the fermented soybean paste and mung bean extract was higher.
Accordingly, the combined extract can provide a preventive or ameliorative effect of arthritis caused by IL-1β including Omija extract, and can be provided as a food or a functional food which is highly palatable and easy to supply.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Of the right.
Claims (8)
For the above Omiza extract
Bacillus herbaceous extract,
Extract of royal jelly
Mixed mung bean extract
A composition for preventing or ameliorating arthritis caused by IL-1 ?.
Wherein the Schizandra chinensis extract is extracted with a solvent selected from the group consisting of water, an alcohol having 1 to 6 carbon atoms and a mixed solvent thereof,
A composition for preventing or ameliorating arthritis caused by IL-1 ?.
Wherein the arthritis is osteoarthritis or rheumatoid arthritis
A composition for preventing or ameliorating arthritis caused by IL-1 ?.
The above-mentioned Omija is a seed of Omija
A composition for preventing or ameliorating arthritis caused by IL-1 ?.
Food composition.
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| KR20200085114A (en) * | 2019-01-04 | 2020-07-14 | 박병희 | A cosmetic composition for improving skin barrier function power containing an extract of fermented omija as an effective ingredient |
| KR20200085126A (en) * | 2019-01-04 | 2020-07-14 | 박병희 | A cosmetic composition for preventing or improving skin wrinkles containing an extract of fermented omija as an effective ingredient |
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| WO2019177327A1 (en) * | 2018-03-12 | 2019-09-19 | 한국 한의학 연구원 | Composition comprising schisandra chinensis extract as effective ingredient for prevention, alleviation, or treatment of arthritis |
| KR102298390B1 (en) * | 2020-12-02 | 2021-09-03 | 동의대학교 산학협력단 | Composition for tissue repair that contains hyaluronic acid and exhibits excellent skin repair effect |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20200085114A (en) * | 2019-01-04 | 2020-07-14 | 박병희 | A cosmetic composition for improving skin barrier function power containing an extract of fermented omija as an effective ingredient |
| KR20200085126A (en) * | 2019-01-04 | 2020-07-14 | 박병희 | A cosmetic composition for preventing or improving skin wrinkles containing an extract of fermented omija as an effective ingredient |
| KR102145030B1 (en) * | 2019-01-04 | 2020-08-14 | 박병희 | A cosmetic composition for improving skin barrier function power containing an extract of fermented omija as an effective ingredient |
| KR102163015B1 (en) * | 2019-01-04 | 2020-10-07 | 박병희 | A cosmetic composition for preventing or improving skin wrinkles containing an extract of fermented omija as an effective ingredient |
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