KR20140129492A - Compositions Comprising a Leaf Extract of Cudrania tricuspidata for the Prevention and Treatment of Arthritis disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition or a health functional food for treating arthritis disease comprising a Cudrania tricuspidata leaf extract as an active ingredient. As a measurement result of effects of expression of inflammatory-related gene and generation of inflammatory cytokine according to hot water extract treatment of Cudrania tricuspidata leaf in a damage experimental model of a cartilage cell derived from H_2O_2 and LPS treatment in the cartilage cell obtained in the joint of a rat, provided is a pharmaceutical composition or a health functional food useful for treating arthritis disease by checking inhibition of expression of inflammatory-related gene and damage of the cartilage cell in damage of the cartilage cell induced by H_2O_2 treatment.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating arthritis, which comprises an extract of Cryptomeria japonica as an active ingredient. The present invention relates to a composition for preventing or treating arthritis,

The present invention relates to a composition for the prevention or treatment of arthritis, which comprises an extract of Lilium japonica as an active ingredient.

[1] Korea National Health and Nutrition Examination Survey. Ministry of Health and Welfare. Seoul, Korea. p 51 (2001), Senior Statistical Reports. The Statistics Korea. Daejeon, Korea. p 5 (2010)

[Literature 2] Garner BC et al., Using animal models in osteoarthritis biomarker research. J Knee Surg 24: 251-264 (2011)

[Literature 3] Wenjun Wu et al., Therapeutic Effect of the Saponin Fraction from Clematis chinensis Osbeck Roots on Osteoarthritis Induced by Monosodium Iodoacetate through Protecting Articular Cartilage. phytother. Res 10: 1002 / ptr. 2977 (2009), Wesche-Soldato DE et al., The apoptotic pathway as a therapeutic target in sepsis. Curr Drug Targets 8: 493-500 (2007),

[Literature 4] Herrington C et al., Molecular and cellular themes of inflammation and immunology. J Pathol 214: 123-125 (2008), Giuseppe MC et al., Glycosaminoglycans Modulate Inflammation and Apoptosis in LPS-Treated Chondrocytes. J Cellular Biochemistry 160: 83-92 (2009)

[Literature 5] Thanyaluck P et al., The effects of p-hydroxycinnamaldehyde from Alpinia galanga extracts on human chondrocytes. Phytochemistry 7: 237-243 (2009)

[Literature 6] MJ Janusz et al., Modulation of iodoacetate-induced experimental osteoarthritis in rats by matrix metalloproteinase inhibitors. Osteoarthritis and cartilage . 9: 751-760 (2001), Okadaa et al., Progress of research in osteoarthritis. Metalloproteinases in osteoarthritis. Clin Calcium 19: 1593-1601 (2009)

[7] Choi SR et al., Antioxidant activity of methanol extracts from Curdrania tricuspidata . Korean J Med Crop Sci 17: 115-120 (2009)

[Literature 8] Kang DG et al., Effects of Cudrania tricuspidata water extract on blood pressure and renal functions in NO-dependent hypertension. Life Sci 70: 2599-609 (2002)

[Literature 9] MJ Janusz et al., Modulation of iodoacetate-induced experimental osteoarthritis in rats by matrix metalloproteinase inhibitors. Osteoarthritis and cartilage . 9: 751-760 (2001),

[10] Okada A et al., Progress of research in osteoarthritis. Metalloproteinases in osteoarthritis. Clin Calcium 19: 1593-1601 (2009)

[Literature 11] Wesche-Soldato DE et al., The apoptotic pathway as a therapeutic target in sepsis. Curr Drug Targets 8: 493-500 (2007)

[Literature 12] Thanyaluck P et al, The effects of p-hydroxycinnamaldehyde from Alpinia galanga extracts on human chodrocytes. Phytochemistry 7: 237-243 (2009)

[Literature 13] Y Yoshihara et al., Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial fluids from patients with rheumatoid arthritis or osteoarthritis. Ann Rheum Dis 59: 455-461 (2000)

[14] MJ Janusz et al., Modulation of iodoacetate-induced experimental osteoarthritis in rats by matrix metalloproteinase inhibitors. Osteoarthritis and cartilage 9: 751-760 (2001)

[Literature 15] Combe R et al., The monosodium iodoacetate model of osteoarthritis; a model of chronic nociceptive pain in rats. Neurosci . Lett . 370: 236-240 (2004)

[16] Lim YM et al., Antioxidant effect of crataegi fructus extract on oxidative stress of reactive oxygen species in cultured human skin fibroblasts. Korean J Oriental Phsiology & Pathology 22: 115-119 (2008)

[Literature 17] Herrington et al., Molecular and cellular themes of inflammation and immunology. J Pathol 214: 123-125 (2008)

[Literature 18] Giuseppe MC et al., Glycosaminoglycans modulate inflammation and apoptosis in LPS-treated chondrocytes. J Cellular Biochemistry 160: 83-92 (2009)

[Literature 19] Amin AR et al., Nitric oxide synthase and cyclooxygenases: distribution, regulation, and intervention in arthritis. Curr Opin Rheumatol 11: 202-209 (1999)

[Literature 20] Asada S et al., Effect of hydrogen peroxide on the metabolism of articular chondrocytes. Inflamm Res. , 48: 399-403 (1999)

The present invention relates to a composition for the treatment and prevention of arthritis, which comprises a leaf extract of Liliaceae.

According to the 1998 and 2001 National Health and Nutrition Surveys, arthritis was a common chronic disease in people over 45 years of age. The prevalence of arthritis increased with age, from 356.7 in 1998 to 364.2 in 2001. According to the recent statistics of elderly people in 2010, the proportion of elderly people aged 65 or older in Korea is 11%, and the prevalence of chronic degenerative diseases is increasing with the aging of population structure, and arthritis accounts for more than 43.1% (2001), Senior Statistical Reports, The Statistics Korea, Daejeon, Korea, p 5 (2010)).

Arthritis is inflammation of the joints, often accompanied by pain, stiffness, and edema, which are caused by degenerative changes, immune system disorders, infection, trauma, metabolic disorders, and more than 100 types (Garner BC et al., Using animal models in osteoarthritis biomarker research. J Knee Surg 24: 251-264 (2011)). Of these, osteoarthritis and rheumatoid arthritis (RA) arthritis account for 80% of all arthritis and are the most burdened by musculoskeletal disorders.

Protein hydrolysis enzymes, cytokines, and nitric oxide (NO) are known to be major factors in the development of degenerative arthritis (Wenjun Wu et al., Therapeutic Effect of the Saponin Fraction from Clematis chinensis Osbeck Roots on Osteoarthritis Induced by Monosodium Iodoacetate through Protecting Articular Cartilage. phytother. Res 10: 1002 / ptr. 2977 (2009), Wesche-Soldato DE et al., The apoptotic pathway as a therapeutic target in sepsis. Curr Drug Targets 8: 493-500 (2007), Herrington C et al., Molecular and cellular themes of inflammation and immunology. J Pathol 214: 123-125 (2008), Giuseppe MC et al., Glycosaminoglycans Modulate Inflammation and Apoptosis in LPS-Treated Chondrocytes. J Cellular Biochemistry 160: 83-92 (2009)). These components constitute cartilage tissue of the joints in particular, and are typical factors affecting the metabolism of chondrocytes.

The metabolism process in cartilage tissue plays a pivotal role in the degeneration of cartilage tissue (Thanyaluck P et al., Phytochemistry 7: 237-243 (2009)), the effects of p-hydroxycinnamaldehyde from Alpinia galanga extracts on human chondrocytes, Typically, matrix metalloproteinases are proteolytic enzymes that degrade bone, cartilage matrix components and chondrocytes and are known to play a major role in pathogenesis such as arthritis (MJ Janusz et al., Moderation of iodoacetate -induced experimental osteoarthritis in rats by matrix metalloproteinase inhibitors. Osteoarthritis and cartilage . 9: 751-760 (2001), Okadaa et al., Progress of research in osteoarthritis. Metalloproteinases in osteoarthritis. Clin Calcium 19: 1593-1601 (2009)).

(IL-1) and tumor necrosis factor (TNF) -alpha in arthritis are differentiated from the cartilage by cartilage It is known as a cytokine that affects catabolism of cell tissue. Most inflammatory reactions are known to involve Cox-2, inducible nitric oxide (iNOS) and inflammatory cytokines such as IL-1β, IL-6, and TNF-α. In addition, it is known that osteoarthritis is caused by overexpression of TNF-α and Cox-2 due to a series of inflammatory reactions caused by macrophages in the joints. (Wesche-Soldato DE et al., The apoptotic pathway as a therapeutic target sepsis. Curr Drug Targets 8: 493-500 (2007)).

Normally produced by iNOS, NO plays an important role in the primary immune defense system and plays an important role in physiological regulation such as vasodilation. However, when iNOS is produced by external factors such as LPS and TNF-α, the production of excessive NO leads to inflammation and cell necrosis. In addition, NO increases the vasodilatation and penetration of joints in the development of degenerative arthritis, which increases the penetration of TNF-α and IL-β from leukocytes into joints and induces apoptosis (programmed cell death) of cartilage cells do. (Thanyaluck P et al., The effects of p-hydroxycinnamaldehyde from Alpinia galanga extracts on human chodrocytes. Phytochemistry 7: 237-243 (2009)).

There are two types of Cox enzymes, Cox-1 and Cox-2, that promote inflammation by promoting the production of Prostaglandin E ₂ (PGE ₂ ). Cox-1 is constantly involved in the synthesis of several types of prostaglandins and is involved in plasma homeostasis, gastric juice secretion, platelet coagulation, etc. In contrast, Cox-2 is temporarily inhibited by external factors such as TNF-α and LPS (Y Yoshihara et al.), Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial fluids from patients with rheumatoid arthritis or osteoarthritis. Ann . Rheum Dis 59: 455-461 (2000)).

It is recognized that maintaining the physiological physiological function of cartilage tissue is due to a balance of tissue anabolism and catabolism. In addition to collagen, tissue inhibitors of metalloproteinases (TIMPs) and proteoglycans (aggrecan), metalloproteinases (MMPs) and nitric oxide NO), TNF-a, collagenase, and aggrecanase. (MJ Janusz et al., Modulation of iodoacetate-induced experimental osteoarthritis in rats by matrix metalloproteinase inhibitors. Osteoarthritis and cartilage 9: 751-760 (2001)).

Cudrania tricuspidata ( Cudrania tricuspidata ) is a deciduous broad-leaved arboreous tree belonging to the family Molaceae. It is distributed in East and South Jeolla provinces, Gyeongsang province, and Chungcheongnam-do province. Recently, antioxidant activities of cucurbit tree Tricuspidata Border according to harvesting parts and time. Korean J Med Crop Sci 17: 115-120 (2009)), antidiabetics, hypertension inhibition (Kang DG et al., Effects of Cudrania tricuspidata water extract on blood pressure and renal functions in NO-dependent hypertension. Life Sci. 70: 2599-609 (2002)). Such physiological activity seems to be attributed to the physiologically active components of the phenolic compounds in the cucurbit tree, Is increasing.

In recent years, the preference and demand of health functional foods for arthritis have increased rapidly in line with the aging society. In the majority of cases, glucosamine and chondroitin are forming marketability. However, some medicines have raised questions about the effect of glucosamine as a functional food. In order to maintain the market of the arthritis health functional food in the future, there is an increasing interest in the noodles at a time when a substitute measure is needed.

However, none of the published literature, cited herein only as a reference, discloses or teaches the therapeutic effects of arthritis of Leaf extract.

The present inventors sought to develop a safe material, particularly a plant-derived material, capable of effectively preventing or treating osteoarthritis. As a result, the extract of Codonopsis thunbergii extracts were added to cartilage cells obtained from rat joints with H ₂ O ₂ And LPS-treated chondrocytes a result of damage to the experimental model measured the effects of inflammatory gene expression and inflammatory cytokine production according to kkujippong leaf extract treated at induction with inflammation in cartilage cell damage induced by H ₂ O ₂ treatment The inventors of the present invention have completed the present invention by confirming that the expression of the related gene and the damage of the cartilage cell are inhibited, thereby protecting cartilage cells in the joint region and inhibiting the inflammatory reaction, thereby being effective for preventing or treating arthritis.

According to one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating osteoarthritis, comprising an extract of Lilium japonica as an active ingredient.

The cucurbitaceae as defined herein include Korean, Chinese, and Japanese, preferably Korean, and more preferably, Jeollanam-do.

The extracts as defined herein include water, alcohol, lower alcohol such as methanol, ethanol, butanol, or a mixed solvent thereof, preferably water-soluble extract.

The osteoarthritis, as defined herein, is useful for treating osteoarthritis, rheumatoid arthritis, Lupus arthritis, chronic active joint inflammation, acute arthritis, an activity disorder associated with nonsteroidal antiinflammatory drugs (NSAIDs), morphological changes due to joint destruction, Osteoarthritis, rheumatoid arthritis, Lupus arthritis, arthritis selected from the group consisting of inflammatory manifestations of joint inflammation, infection and systemic inflammatory disease due to reduced immunity due to stress.

Specifically, it is characterized by being caused by at least one cause selected from the group consisting of the aforementioned physical stress, mental stress, oxidative stress, nonsteroidal antiinflammatory drug, steroid preparation, and inflammatory disease caused by immunity depression .

Hereinafter, a production method for obtaining the extract of the present invention will be described in detail.

For example, the extract of the present invention may be prepared by washing and drying the leaf of a tree, then washing the leaf with water, alcohol, C ₁ to C ₄ lower alcohols or a mixed solvent thereof, preferably water or a mixed solvent of water and ethanol, more preferably water as an extraction solvent, at a temperature of about 10 to 120 ° C, preferably 20 to 110 ° C Preferably 1 to 10 times by a conventional extraction method such as room temperature extraction method, heat extraction method, ultrasonic extraction method and reflux extraction method, preferably reflux extraction method, at a reaction temperature for about 30 minutes to 6 days, preferably 1 hour to 48 hours, A second step of repeating extraction 2 to 7 times; A third step of filtrating and concentrating the extract obtained in the above step under reduced pressure; And a fourth step of lyophilizing the concentrated extract at about -50 ° C to -30 ° C for about 24 to 72 hours to obtain the extract of Cucurbitaceae of the present invention.

Accordingly, the present invention provides a pharmaceutical composition and a health functional food for the prevention and treatment of arthritis containing the extract of Cucurbitaceae as an active ingredient, obtained by the above-mentioned production method and the above-mentioned production method.

In the experimental model of chondrocyte injury induced by H ₂ O ₂ and LPS treatment on the cartilage cells obtained from the joints of rats for the hot-water extract of the present invention, inflammation-related gene expression and inflammatory cytokine production As a result, it was confirmed that inflammation-related gene expression and chondrocyte damage were suppressed in the cartilage cell damage induced by H ₂ O ₂ treatment, thereby being useful as a composition and health functional food for preventing and treating arthritis Respectively.

The composition of the present invention contains the herbal extract in an amount of 0.1 to 50% by weight based on the total weight of the composition.

However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

Compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The composition containing the extract according to the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions, Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract is preferably administered at a dose of 0.01 mg / kg to 10 g / kg per day, preferably 1 mg / kg to 1 g / kg per day. The administration may be carried out once a day or divided into several doses. Therefore, the dose is not intended to limit the scope of the present invention in any aspect.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, including, for example, oral and rectal, or intravenous.

The present invention also provides a health functional food for prevention and improvement of arthritis containing an extract of Lilium japonica as an active ingredient.

The health functional food containing the extract of the present invention can be used variously for medicines, foods and beverages for prevention and improvement of arthritis. Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, health supplements and the like, and they can be used as powders, granules, tablets, capsules or beverages have.

Accordingly, the present invention also provides a food and food additive containing, as an active ingredient, Cucurbitacean leaf extract having an effect of preventing and improving arthritis.

The food forms to which the extract of the present invention can be added include various foods of candy, beverages, gums, tea, vitamin complexes, or foods that are health supplement foods.

The extract of the present invention can be added to foods or beverages for the purpose of preventing and improving arthritis. At this time, the amount of the extract in the food or beverage is generally 0.01 to 15% by weight of the total food weight of the health food composition of the present invention, and the health beverage composition is 0.02 to 10 g based on 100 ml, Can be added at a ratio of 0.3 to 1 g.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient, such as ordinary beverages, in addition to containing a mixture of the above extract as an essential ingredient in the indicated ratios, have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention relates to a pharmaceutical composition for the treatment of arthritis or a health functional food containing the extract of Lilium japonica leaf as an active ingredient, wherein the extract is used for treating cartilage cells obtained from the joints of rats by H ₂ O ₂ and LPS- In the experimental model, inflammation - related gene expression and inflammatory cytokine production were evaluated by hydrothermal extracts from the leaves of Cucumis japonica. The results showed that inflammation - related gene expression and cartilage cell damage were observed in H ₂ O _{2 -} It is effective to treat arthritis.

1 is a process of obtaining chondrocytes from the cartilage region of Rat by primary culture;
FIG. 2 is a cytotoxicity test result of leaf hot-water extract from chrysanthemum cells;
FIG. 3 shows the result of measuring the protective effect of hydrolyzed leaf extract of Cucumber leaf after inducing oxidative damage by treating H ₂ O ₂ with chondrocytes;
FIG. 4 shows the results of measurement of the expression level of inflammation-related cytokines in order to examine the effect of reducing the inflammatory response by treatment with hot-water extracts of chrysanthemum morifolium in chondrocytes;
FIG. 5 shows the results of measurement of the expression level of nitric oxide (NO) by active lipase (ROS) induced by LPS treatment on chondrocytes in order to examine the effect of reducing the inflammatory response by treatment with hot water extract ;
FIG. 6 is a graph showing the results of measuring the expression of PGE ₂ , an inflammatory expression factor, after inducing inflammation by treating LPS in chondrocytes in order to investigate the effect of reducing the inflammatory response by treatment with hot-water extract of Cucurbitaceae in chondrocytes;
FIG. 7 is a graph showing the degree of expression of anabolism and catabolism factors involved in physiological and physiological actions of chondrocytes after inducing oxidative damage by treating H ₂ O ₂ with chondrocytes .

Hereinafter, the present invention will be described in detail with reference to the following Reference Examples and Experimental Examples.

However, the following Reference Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Reference Examples and Experimental Examples.

Example  One. CJ  Manufacture of tree leaf extract

Cucumber leaves are domestic products purchased in June, 2012 Jeonnam herbal medicine (243-5, Hwasun-eup, Hwasun-eup, Jeonnam). Dried and supplied, were crushed with a crusher (Hanil, Korea) and used. 1,000 mL of distilled water was added to 50 g of the sample, and the mixture was heated at 100 ° C for 2 hours and 30 minutes. The extract was concentrated under reduced pressure using a rotary vacuum evaporator (Sunil EYELA, Rotatory evaporator, N-1N). Then, the lyophilized extract (6 g) was obtained by freeze-drying and used as a sample in the following experimental examples (hereinafter referred to as "CTW").

Experimental Example  1. Experimental model of arthritis using chondrocytes obtained from animals

In order to confirm the efficacy of the sample obtained in the above example for arthritis using chondrocytes, an experiment was conducted by applying the method described in the literature (Combe R et al., The monosodium iodoacetate model of osteoarthritis: a model of chronic nociceptive pain in rats. Neurosci . Lett . 370: 236-240 (2004)).

Chondrocyte cells obtained from SD rats used in this test are suitable cells for efficacy evaluation and widely used in osteoarthritis experimental models at the cellular level and have accumulated abundant test basic data and can use these data for analysis and evaluation of test results .

Chondrocytes for experiment were obtained from the primary culture of rats, and the progress of primary culture proceeded as follows.

The experimental animals for cartilage harvesting were anesthetized with male Sprague-Dawley rats weighing about 180-200 g, 4 to 6 weeks old, supplied from Korea BioLink, and disinfected with 70% alcohol after cervical dislocation. Were collected in sizes of about 2 to 3 mm. The collected cartilage tissues were stored in phosphate buffered saline (HClone SH30256.01) solution, transferred to a clean bench, and incubated for 30 min in 0.1% EDTA-CDMF (Sigma E5134) ) were _{(37 ℃, 5% CO 2} ). The cells were incubated with 0.25% trypsin for 1 hour (37 ° C, 5% CO ₂ ) and overgrafted overnight at 2 mg / ml collagenase type I (Sigma, C0130). The cell suspension was filtered through a 100-μm pore size cell strainer (BD Falcon, 352360, USA), centrifuged at 1,600 rpm for 10 minutes, centrifuged and washed three times with Hank's balanced salt solution (Hyclone SH30031.01) After that, chondrocytes were obtained. The cells were washed with 1% penicillin-Streptomycin (Hyclone, SV30010) and 1% L-glutamine (Hyclone, SH30034.01) in DMEM (Hyclone, SH30243.01) with 10% FBS (Fetal Bovine Serum, And cultured in a 75T flask with 0.1% gentamycin sulfate (Lonza 17-518Z, USA) (FIG. 1).

Experimental Example  2. Cell viability ( Cell Viability ) Measure

In order to confirm the effect on the cell survival rate using the chondrocyte of the sample obtained in the above examples, an experiment was conducted by applying the method described in the literature (Lim YM et al., Antioxidant effect of crataegi fructus extract on the oxidative stress of reactive oxygen species in cultured human skin fibroblasts. Korean J Oriental Phsiology & Pathology 22: 115-119 (2008)).

The chondrocytes obtained from the primary culture were subcultured in a DMEM culture medium at a concentration of 5 × 10 ⁴ cells / well in a 96-well plate and stabilized overnight. Then, the extract dissolved in the third distilled water was treated for 24 hours (toxicity test) or 2 hours (H ₂ O ₂ Lt; / RTI >

20 μl of 3- (4,5-methylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT, Sigma M5655) reagent was treated for up to 4 hours at 37 ° C in an incubator (Thermo scientific, Heraeus BB15) The post-culture medium and the MTT reagent were removed, and 200 μl of DMSO reagent was added thereto, and measurement was carried out at 560 nm using an ELISA reader (VERSAMAXSL-20 Molecular Devices, Korea). The mean value and the standard error were obtained by three measurements.

As a result of the experiment, the cytotoxicity test was carried out at a concentration of 1000 ㎍ / mL at a maximum concentration of the leaf water extract of Cjjimpon leaf, and no cytotoxicity was observed at all concentrations (FIG. 2). In addition, H ₂ O ₂ As a result of observing the protective effect against oxidative damage induced by 700 treatment, cell viability was 46.39 ± 15.14 in cells treated with H ₂ O ₂ compared with untreated chondrocytes (100). In contrast, the cell survival rate was increased to 62.49 ± 4.82 at the concentration of 200 ㎍ / mL of coconut leaf water extract, indicating that the protective effect of chondrocytes was exhibited. In addition, at higher concentrations, the effect decreased and the cell viability decreased (FIG. 3).

Experimental Example  3. ELISA  By method Cytokine ( cytokines ) Measure

In order to confirm the effect of the ELISA method on the cytokines of the samples obtained in the above examples, experiments were conducted as described below (Herrington et al., Molecular and cellular themes in inflammation and immunology. J Pathol 214: 123-125 (2008)).

Osteoarthritis is a disease in which the cartilage of the joint wears and causes inflammation accompanied by pain in its vicinity. The inflammatory reaction of osteoarthritis secretes TNF-alpha and IL1-beta, which are cytokines related to osteoarthritis, around the joints. These cytokines again cause inflammation reaction in the joints to secrete NO, PGE2, etc., . In vitro In the experiment, proinflammatory cytokines can be induced by treating Lipopolysaccharide (LPS), which is an inflammatory substance in cartilage cells. In order to confirm the expression of TNF-alpha and IL1-beta, one of the proinflammatory cytokines, the chondrocytes obtained from the primary culture were dispensed into DMEM culture medium at a concentration of 5 × 10 ⁴ cells / well on a 96-well plate, After incubation for 24 hours with samples and LPS (50 μg / mL), two cytokines were measured after supernatant. (Rat TNF-alpha; DY510, Rat IL-1beta; DY501) was performed using the ELISA protocol of R & D Systems and measured using an ELISA reader (Molecular Devices, Korea).

In this experiment, high-level LPS treatment was required for the characteristics of cartilage tissue cells. Experimental results showed that optimal inhibition of TNF-α and IL-1β production was observed at 50 μg / ml (data not shown). First, TNF-α expression was highest in LPS-treated chondrocytes at 194.2 ± 38.83, and significantly decreased to 87.83 ± 27.84 at 200 μg / mL of Cryptomeria japonica leaf. Respectively. It was also confirmed that the expression of IL-1β was also significantly reduced in cells treated with cucurbit leaf, and in this case, the expression was decreased at all the treatments (FIG. 4).

Experimental Example  4. LPS Induced by Nitric oxide  ( NO ) Production inhibition measurement

In order to confirm the effect of LPS-induced nitric oxide (NO) production of the samples obtained in the above-mentioned Examples, the experiment described in the literature was applied as follows (Giuseppe MC et al., Glycosaminoglycans modulate inflammation and apoptosis in LPS-treated chondrocytes. J Cellular Biochemistry 160: 83-92 (2009)).

For the NO determination, the chondrocytes obtained from the primary culture were inoculated into a 96-well plate at a concentration of 5 × 10 ⁴ cells / well in a DMEM culture medium, stabilized overnight, treated with samples and LPS (50 μg / The supernatant was measured. Collect 100 μL of the supernatant in a new 96-well plate, add 100 μL / well of Griess reagent (Sigma), and measure the absorbance at 570 nm.

As a result, NO production in LPS-treated chondrocytes was 100%, whereas NO expression was reduced to 80% in chondrocyte treated with 100 μg / (Fig. 5).

Experimental Example  5. LPS Induced by PGE2 Production inhibition measurement

In order to confirm the effect of LPS-induced production of PGE2 on the samples obtained in the above examples, experiments were conducted as described below (Amin AR et al., Nitric Oxide Synthase and cyclooxygenases: distribution, regulation, and intervention in arthritis. Curr Opin Rheumatol 11: 202-209 (1999)).

In order to confirm expression of PGE2, chondrocytes obtained from the primary culture were dispensed into DMEM culture medium at a concentration of 5 × 10 ⁴ cells / well on a 96-well plate and stabilized overnight. Then, samples and LPS (50 μg / mL) After culturing for 24 hours, the supernatant was taken and PGE2 was measured. For the measurement, an R & D PGE2 ELISA kit (R & D Systems, KGE004B) was used and experiments were conducted according to the General ELISA protocol and measured using an ELISA reader (Molecular Devices, Korea).

The experimental results, in chondrocytes treated with kkujippong leaves 200 μg / mL compared to cartilage treated only with LPS cells PGE ₂ The expression was observed to decrease, but did not show any significant difference (Fig. 6).

Experimental Example  6. H ₂ O ₂  Depending on the treatment Oxidative  Measurement of related gene expression by damage

In order to confirm the effect on the expression of the related genetic elements by the oxidative damage according to the H ₂ O ₂ treatment of the samples obtained in the above examples, experiments were carried out by applying the method described in the literature (Asada S et al , Influence of hydrogen peroxide in the metabolism of articular chondrocytes. Inflamm Res, 48: 399-403 (1999)).

Divides the cartilage cells separated from experimental animals in the 6-well plate DMEM culture medium supplemented with 10% FBS at a concentration of 5x10 ⁶ cells / well in after stabilization for 12 hours, the cuts of kkujippong ethanol extract with H ₂ O ₂ Total RNA was extracted with RNeasy extraction kit (QIAGEN, Maryland, USA) using 0.25% trypsin-EDTA for 2 hours. The cDNA was synthesized by iScript Select cDNA Synthesis 4 μL of 5 × iScript select reaction mix, 2 μL of Oligo (dT) Primer set (see Table 1), 5 μl of RNA sample, and 5 μL of Nuclease-Free Water And 1 μL of iScript Reverse Transcriptase was added to each well and pipetted up and down to mix well. After reacting at 42 ° C for 60 minutes and 85 ° C for 5 minutes, the synthesized cDNA was used for PCR reaction. The Rael-Time PCR was performed using the Step One Real-Time PCR System (Applied Biosystems, USA) and protocol of iQ SYBR Green Supermix (BIO-RAD, BIO-RAD, Singapore). The final concentration of the mixture for PCR was 2 μl (10-100 ng) of cDNA, 10 μl of 2X iQ SYBR Green supermix, 1 μl (250 nM) of each forward and reverse primer, and 7 μl of H ₂ O. PCR was carried out at 95 ° C for 10 minutes, followed by 15 cycles at 95 ° C for 15 seconds, 55 ° C for 15 seconds, and 72 ° C for 30 seconds, followed by 15 cycles of 95 ° C for 15 seconds, 60 ° C for 1 minute, After 15 seconds of polishing step, PCR analysis was performed. The primers used in the reactions are shown in Table 1 (Table 1).

Primer set sequence used for Real - Time PCR Sequence
Name Forward Sequence Reverse Sequence NCBI
Reference GAPDH TGG CCT CCA AGG AGT AAG AAA C CAG CAA CTG AGG GCC TCT CT BC087743.1 Aggrecan GAA GTG GCG TCC AAA CCAA CGT TCC ATT CAC CCC TCTCA NM_022190.1 Collagen Type  I GAG CGG AGA GTA CTG GAT CGA CTG ACC TGT CTC CAT GTT GCA BC133728 Collagen Type II GCA ACA GCA GGT TCA CGT ACA TCG GTA CTC GAT GAT GGT CTT G L48440 TIMP -One AAG GGC TAC CAG AGC GATCA ATC GAG ACC CCA AGG TAT TGC NM_053819.1 TIMP -3 GAC CGA CAT GCT CTC CAA TTT C GCT GCA GTA GCC ACC CT CT U27201.1 MMP -3 GAG TGT GGA TTC TGC CAT TGA G TTA TGT CAG CCT CTC CTT CAG AGA NM_133523.2 MMP -7 ACT CTA GGC CAT GCC TTT GC CCA TCC GTC CAG TAC TCA TCC T NM_012864.2

In this experiment, collagen type I, identified as an anabolic factor, is most commonly distributed in human cells as well as chondrocytes. Collagen type II accounts for 50% of the cartilage tissue proteins and among the collagen types distributed in cartilage It is a protein that occupies 85-90%. Therefore, the formation of collagen types I and II is an important determinant of cartilage tissue status. In this experiment, the treatment of cartilage tissue cells with 700 μM H ₂ O ₂ was found to significantly reduce the expression of collagen type I gene. Based on the results of previous experiments, gene expression experiments were carried out with a maximum leaf concentration of 200 μg / mL.

As a result, collagen expression was reduced by 90% in cells treated with 700 μM of H ₂ O ₂ compared to normal cells, and collagen type I expression was 50% and collagen type II was 140% at a concentration of 200 μg / mL , And significantly increased collagen expression was observed. Aggrecan is one of the representative proteoglycan core proteins synthesized in cartilage tissue. It is known that it acts to maintain joints by showing resistance from external pressure (compressive load). The expression of Aggrecan gene was significantly higher than that of cells treated with 700 H ₂ O ₂ alone, indicating that aggrecan gene expression was significantly increased in cells treated with selected sources, (Fig. 7).

Experimental Example 7. Single-dose toxicity test

Single - dose toxicity studies were performed on the leaf extracts of. As a result of the single dose toxicity test, it was not possible to observe the 4-week death at 2 g / kg, which is the dose of ICH established in the case of CJP leaf extract, and we found no significant abnormality in weight gain and feed intake There was no. Therefore, it was found that the extract of the present invention is a safe drug.

Hereinafter, formulation examples of the composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Preparation Example 1. Preparation of powder

CTW extract ------------------------------------------- 20 mg

Lactose ------------------------------------------------ 100 mg

Talc ------------------------------------------------- 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

CTW extract ------------------------------------------- 10 mg

Corn starch ------------------------------------------ 100 mg

Lactose ------------------------------------------------ 100 mg

Magnesium stearate ------------------------------------- 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

CTW extract ------------------------------------------- 10 mg

Crystalline cellulose - 3 mg

Lactose ------------------------------------------- 14.8 mg

Magnesium Stearate ------------------------------- 0.2 mg

The above components are mixed in accordance with a conventional method for producing a capsule, and filled in a gelatin capsule to prepare a capsule.

Formulation example  4. Preparation of injections

CTW extract ------------------------------------------- 10 mg

Mannitol ---------------------------------------------- 180 mg

Sterile sterile distilled water for injection --------------------------------- 2974 mg

Na ₂ HPO _{4 ,} 12H ₂ O ----------------------------------------- 26 mg

(2 ml) per ampoule in accordance with the usual injection method.

Formulation example  5. Liquid  Produce

CTW extract ------------------------------------------- 20 mg

Isolation Party ---------------------------------------------- 10 g

Mannitol ------------------------------------------------- 5 g

Purified water ------------------------------------------------

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Formulation example  6. Manufacture of health food

CTW extract ------------------------------------------- 1000 mg

Vitamin mixture -------------------------------------------

Vitamin A Acetate ------------------------------------ 70 g

Vitamin E ---------------------------------------------- 1.0 mg

Vitamin B1 -------------------------------------------- 0.13 mg

Vitamin B2 -------------------------------------------- 0.15 mg

Vitamin B6 --------------------------------------------- 0.5 mg

Vitamin B12 -------------------------------------------- 0.2 g

Vitamin C ----------------------------------------------- 10 Mg

Biotin ------------------------------------------------- 10 [mu] g

Nicotinic acid amide 1.7 mg

Folic acid ------------------------------------------------- - 50 [mu] g

Calcium pantothenate ----------------------------------------- 0.5 mg

Inorganic mixture -------------------------------------------

Ferrous sulfate 1.75 mg < RTI ID = 0.0 >

Zinc oxide - 0.82 mg

Magnesium carbonate ----------------------------------------- 25.3 mg

Potassium phosphate monohydrate 15 mg

Secondary calcium phosphate -------------------------------------------- 55 mg

Potassium citrate --------------------------------------------- 90 mg

Calcium carbonate ---------------------------------------------- 100 mg

Magnesium chloride ----------------------------------------- 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7. Manufacture of health drinks

CTW extract ----------------------------------------- 1000 mg

Citric acid --------------------------------------------- 1000 mg

Oligosaccharide --------------------------------------------- 100 g

Plum concentrate --------------------------------------------- 2 g

Taurine ------------------------------------------------- 1 g

Add purified water - 900 ml total

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated for about 1 hour at 85 DEG C with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, ≪ / RTI >

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (9)

A pharmaceutical composition for preventing or treating arthritis comprising an extract of Cudrania tricuspidata Leaves as an active ingredient. The pharmaceutical composition according to claim 1, wherein the extract is an extract soluble in water, alcohol, methanol, ethanol, butanol or a mixed solvent thereof. 2. The method of claim 1 wherein the arthritis is caused by one or more causes selected from the group consisting of physical stress, mental stress, oxidative stress, nonsteroidal antiinflammatory drugs, steroid agents, and inflammatory diseases due to immunosuppression Or a pharmaceutically acceptable salt thereof. The method of claim 1, wherein the arthritis is selected from the group consisting of osteoarthritis, rheumatoid arthritis, Lupus arthritis, chronic active joint inflammation, acute arthritis, an activity disorder associated with nonsteroidal antiinflammatory drug (NSAID) Wherein the inflammatory disease is osteoarthritis selected from the group consisting of inflammation, joint inflammation, infection, and systemic inflammatory disease caused by decreased immunity due to pain, stress, and the like. A health functional food for prevention and improvement of osteoarthritis containing an extract of Curcuma longa as an active ingredient. The health functional food according to claim 5, which is a powder, a granule, a tablet, a capsule or a drink. Food containing cfu tree leaf extract as an active ingredient having the effect of preventing and improving arthritis. [8] The food according to claim 7, wherein the food is candy, a food, a drink, a gum, a tea, a vitamin complex, or a health supplement food. A food additive containing an extract of Cajunpomoe leaf having an effect of preventing and improving arthritis as an active ingredient.

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