KR101607545B1 - Composition for preventing, improving or treating of arthritis comprising herbal extract mixture as effective component - Google Patents

Abstract

The present invention relates to a composition containing a herbal medicine extract mixture as an active ingredient for the prevention, alleviation, or treatment of arthritis, and more specifically, to a composition containing a herbal medicine extract mixture as an active ingredient for the prevention, alleviation, or treatment of arthritis, which can be used as heath functional food or a pharmaceutical composition. According to the present invention, the herbal medicine extract mixture (YNH431) inhibits the expression of ERK, p38 MAPK, and NF-kB caused by the excessive proliferation of synovial fibroblasts, thereby having an inflammation inhibitory effect and an excellent inhibitory effect against the differentiation of osteoclasts associated with a fracture in a joint. It is verified that the expression rate of MMPs, COX-2, and PGE2 increased by inflammation is further reduced in the case of using the herbal medicine extract mixture of the present invention than in the case of separately using each herbal medicine extract. Furthermore, the expression of IL-1β and TNF-α, which are inflammatory cytokines, is inhibited in animal models of inflammatory arthritis, thereby inhibiting the generation of arthritis. Also, an effect of inhibiting a fracture in a joint is shown even in histological and micro-CT findings. Therefore, the herbal medicine extract mixture according to the present invention can be practically used for the composition for the prevention, alleviation, or treatment of arthritis. The composition contains, as an active ingredient, a mixture consisting of 30-50 wt% of a Cudrania tricuspidata stem extract, 20-40 wt% of an Angelica gigas Nakai root extract, 5-15 wt% of a Kalopanax Pictus Nakai stem extract, 5-15 wt% of a Sambucus williamsii HANCE var. coreana Nakai stem extract, 1-10 wt% of a Gastrodia elata Blume root extract, and 1-10 wt% of a Clematis chinensis Osbeck root extract.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing, ameliorating or treating arthritis containing an herbal extract mixture as an active ingredient.

The present invention relates to a composition for preventing, ameliorating or treating arthritis containing an extract of herbal medicine as an active ingredient, and more particularly to a composition for preventing, improving or treating arthritis containing an extract of herbal medicine, Or a composition for preventing, ameliorating or treating osteoarthritis.

Arthritis is a disease that causes inflammation and pain in the joints, and can be divided into osteoarthritis (rheumatoid arthritis) and rheumatoid arthritis (rheumatoid arthritis). Osteoarthritis is sometimes referred to as degenerative arthritis. It occurs predominantly in old age without any specific physical cause. If it progresses chronically, degenerative arthritis is accompanied by movement disorder such as walking disorder. Osteoarthritis is mainly caused by progressive damage or degenerative changes of the cartilage, resulting in damage to the bones and ligaments of the joints, resulting in inflammation and pain. When the osteoarthritis progresses, the production of inflammatory cytokines such as TNF-α and IL-1β (interleukin-1β) is increased, and collagenase, The secretion of matrix metalloproteinases (MMPs), such as stromelysin, is increased, leading to destruction of articular cartilage. In addition, MMPs induce IL-1β, TNF-α and the like, which in turn affect tissues such as muscles, tendons, ligaments, etc., resulting in severe pain. Here, MMPs are the major factors involved in cartilage damage, including collagenases, gelatinases, stromelysins, membrane-type MMPs, and several other MMPs It can be divided into groups.

Rheumatoid arthritis is an inflammatory disease characterized by multiple arthritis, and autoimmunity is known as a major mechanism. Symptoms include inflammation of synovial membrane tissue, macrophage, dendritic cell, T lymphocyte, B lymphocyte, etc., migrate to synovial tissue, resulting in increased joint fluid and joint pain. When inflammation continues, hyperplasia of inflammatory synovial tissue destroys bones and cartilage, deforming the joint structure and causing movement disorder. Studies have shown that inflammatory cytokines in rheumatoid arthritis produce collagenase and protease in synovial fibroblasts and chondrocytes, and these enzymes break down collagen and proteoglycan to produce joints It is known to destroy cartilage. When arthritis occurs, joint inflammation, inflammation, warmth and edema due to inflammation and joint pain are generated, and arthritis symptoms persist and eventually cartilage and bone tissue are destroyed, causing deformation and movement disorder of joint structure. Therefore, there is a desperate situation in which a drug capable of ameliorating or treating movement disorders as chronic symptoms while suppressing inflammation and pain as common symptoms of acute and chronic arthritis is urgent. In addition, there is an urgent need for a drug that has the function of not having toxicity to the cartilage tissue and the synovial tissue, so that the joint structure can be protected and maintained.

Cudrania tricuspidata is a deciduous broad-leaved arboreous tree belonging to the family Molaceae. It is distributed in Jeolla-do, Gyeongsang-do, and Chungcheong provinces in East Asia and Korea. Recently, antioxidant activity of curdrania 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 due to the physiologically active components of the phenolic compounds of the cucurbitaceae. Interest is growing.

Angelica sinensis ) is the angelica of Umbelliferae ( Angelica gigas NAKAI) refers to the dried root, in China, Angelica ( Angelica It used to dry the roots of sinensis DIELS), and the Japanese have been using to dry the roots of ildanggwi (Angelica acutiloba KITAGAWA). As a summary of blood clots, Angelicae has been effectively prescribed for the treatment of maternal postpartum anemia, anemia, headache, etc. (Commercially available medicinal plant illustrations, Jong Hee Park, Jung Kyun Lee, Shin Il Sang, pp409-411 , 2000; Herbal medicine herbal medicine, Health News, pp684-686, 1998).

Pine ( Kalopanax Pictus Nakai) is a deciduous arboreous tree of the Araliaceae family. In spring and summer in herbal medicine, the skin of the stem is peeled off, the skin is peeled off, and it is sun dried and used as medicinal material. From one room to the bark, the muscle is used as a medicament for germane and anal labor. In the private sector, the skin and leaves are used for skin diseases, ulcers, and infectious wounds. (Phytopathological study of Korean Araliaceae, Journal of Plant Taxonomy 19: 103-121, 1989).

Gastrodia elata BLUME) is a thousand miles (Gastrodia the parasitic perennial plants belonging to the Orchidaceae (Orchdaceae) elata BLUME), which is a flattened cylindrical to waterproof type, and has a light yellowish brown color with irregular vertical wrinkles and rounded nodes. It is produced in Gangwon, Gyeonggi province, China and Japan, and has natural acid and artificial cultivated medicines. Chunma has been used for centuries as a traditional herbal medicine in Asian countries, used as an antiepileptic agent, as a stabilizer for analgesic and dizziness, hypertension, general paralysis and tetanus. Gastrodin, phenolic compounds, organic acids, sugars and β-sitosterol are the active ingredients (Chung Byeobo and Shin Min-kyo, Yeonglim Publishing Co., Ltd., pp 138-139, 1998) Vanillyl alcohol and gastrodine have been known to have antiepileptic effects and recently it has been reported that the components of the gum inhibit glutamate-induced apoptosis in neurons (YS Lee, et al., Arch Pharm Res., 22, p404, 1999). In addition, it has been reported that the esterase fraction of the horsetail reduces the decrease of gamma-aminobutylic acid (GABA), increases the glutamate content, and exhibits anti-epileptic effect in pentylene tetrazole-induced epilepsy (JH Ha, et al., J. Endopharmacol., 73, p. 329, 2000).

Sambucus there is williamsii HANCE. coreana NAKAI) is also called the elk tree and the horse pee tree, and is a deciduous tree. The premise of the junction is shown to be sedative when 20 g / kg is injected into the stomach of the rats. Strength is not better than morphine but superior to sulpyrin. After administration, the experimental rat becomes stable. It is effective in treating gout, improving blood circulation and stopping pain. It is known to be effective in the treatment of rheumatoid arthritis, back pain, sprains, fractures, arthritis, neuralgia, edema, gout, nephritis, nervous breakdown, postpartum anemia, ; Daehae spices (herbal medicine) metabolism, Young Lim, pp. 941-942, 1998).

Clematis chinensis Osbeck is a perennial perennial herbaceous plant of Clematis mandshurica var.koreana, which is a herbaceous plant of the dicotyledonous plant. It has the discomfort of joints, limb paralysis, back pain and leg pain , And muscle paralysis. It is also used when the bruise persists. (Ji Hyungjoon, Korean Medicinal Chemistry Association, Korean Medical Index, 290p, 1998).

Korean Patent No. 1373245 discloses a composition for preventing or treating osteoarthritis comprising an extract of natural herbal medicine as an active ingredient and Korean Patent No. 1319866 discloses a composition for preventing or treating osteoarthritis or rheumatoid arthritis and osteoarthritis For the prevention, amelioration or treatment of < RTI ID = 0.0 > a < / RTI > However, there is no mention of a composition for preventing, improving or treating arthritis, which contains as an active ingredient a herbal medicine extractive mixture including Cucumis japonica, Angelica keiskei, Nymphs, Knotted kelp, Chew mackerel and Glycyrrhizae.

Accordingly, the present invention provides a composition for preventing, ameliorating or treating arthritis containing an extract of herbal medicine comprising an extract of a herbal medicine comprising ganoderma lucidum, Angelica keiskei, . The herbal extract mixture of the present invention inhibits the expression of ERK (extracellular signal-regulated kinase), p38 MAPK (mitogen-activated protein kinase) and NF-kB (nuclear factor kappa B) by overexpression of synovial fibroblast And osteoclast differentiation involved in the destruction of joints, and when the herbal extract mixture of the present invention was treated, the MMPs, COX-2 (Cyclooxygenase-2) and PGE2 (Prostaglandin E2). In addition, it inhibits the expression of inflammatory cytokines IL-1β and TNF-α in inflammatory arthritis animal molluscs to inhibit arthritis, inhibits joint destruction in tissue and micro-CT findings, and prevents or improves arthritis The present invention has been completed.

In order to achieve the above object, the present invention provides a health functional food composition for preventing or ameliorating arthritis containing an extract of herbal medicine as an active ingredient.

The present invention also relates to a method for producing a plant extract, which comprises 30 to 50% by weight of a stem extract, 20 to 40% by weight of angelica root extract, 5 to 15% by weight of a stem extract, 5 to 15% % Root extract of Angelica keiskei koidz. And a mixture of 1 to 10 wt.% Of root extract of roast gland root as an active ingredient.

The herbal extract mixture (YNH431) of the present invention inhibits the expression of ERK, p38 MAPK and NF-kB by overexpression of synovial fibroblasts, and thus has excellent inhibitory effect on osteoclast differentiation involved in inflammatory reaction and destruction of joints , And it was confirmed that the expression amounts of MMPs, COX-2 and PGE2 enhanced by inflammation were further reduced when the herbal medicine extract mixture of the present invention was treated than when each herbal medicine extract alone was treated. In addition, it inhibited the expression of inflammatory cytokines IL-1β and TNF-α in an animal model of inflammatory arthritis, inhibiting arthritis, and inhibiting joint destruction in tissue and micro-CT findings. Therefore, the herbal medicine extract mixture according to the present invention can be usefully used in compositions for preventing, improving or treating arthritis.

1 is a graph showing the activity of inhibiting the proliferation of synovial fibroblasts according to the herbal medicine extract mixture treatment of the present invention. (A) inhibited the proliferation of synovial fibroblasts according to the concentration of herbal extract mixture; (B) shows the inhibition of proliferation of synovial fibroblasts by treatment with IL-1β (1.0 ng / ml) and herbal extract mixture (100 μg / ml); YNH431 is a herbal medicine extract mixture containing Cucurbitaceae, Angelica gigas, Pomegranate, Staphylococcus epidermidis, Chimmae, and gestation line.
FIG. 2 shows the results of a comparative analysis of inhibition of signal transduction by treatment of herbal medicine extract mixture, cucumber extract and Angelica gigantosa L. extract in synovial fibroblast cells. (A) is the extract of Euphorbiaceae (EACT); (B) is Angelica gigantosa extract (EAAS); (C) is the result of suppression of signal transduction in synovial fibroblasts treated with herbal extract mixture (YNH431); β-actin is a loading control.
FIG. 3 shows the results of inhibiting the expression of COX-2, MMPs and TIMPs (Tissue inhibitor of metalloproteinase) in the inflammatory reaction of synovial fibroblasts according to the treatment of herbal extract mixture (YNH431) and herbal medicine extract of the present invention. FIG. 3A shows a reverse transcription polymerase chain reaction (RT-PCR) result, and FIG. 3B shows a Western blotting result. Control is a negative control without any treatment; β-actin and GAPDH are loading controls.
FIG. 4 is a graph showing inhibition of the production of PGE2 (Prostaglandin E2) in the inflammatory reaction of synovial fibroblasts according to the treatment of herbal medicine extract mixture (YNH431) and herbal medicine extract of the present invention. Control is a negative control without any treatment.
FIG. 5 shows the results of inhibiting the expression of COX-2, MMPs and TIMPs in the inflammatory reaction of synovial fibroblasts according to the mixing ratio of herbal medicine extracts. NPs are herbal extract mixtures; A scolding stem; The sac is the root of Angelica gigas; Moth is a stem; Folded stalk; Chun means root; Above; β-actin is a loading control.
FIG. 6 shows results of TRAP (Tartrate resistant acid phosphatase) staining for confirming the inhibitory effect of osteoclast differentiation according to the herbal medicine extract mixture treatment of the present invention. (A) shows inhibition of osteoclast differentiation according to the concentration of herbal extract mixture; (B) shows inhibition of osteoclast differentiation by treatment with IL-1β (1.0 ng / ml) and herbal extract mixture (100 μg / ml); YNH431 is a herbal extract mixture.
FIG. 7 shows the inhibitory effect of the osteoclast differentiation according to the herbal medicine extract mixture treatment of the present invention using a resorption pits assay. YNH431 is a herbal extract mixture.
FIG. 8 is a graph showing the degree of inhibition of signal transduction of osteoclast by the herbal extract mixture treatment of the present invention. YNH431 is a herbal extract mixture; β-actin is a loading control; GAPDH is a loading control.
FIG. 9 is a summary of the animal experiment method using the herbal medicine extract mixture of the present invention. YNH431 is a herbal extract mixture.
FIG. 10 shows the inhibitory effect on the occurrence of inflammatory arthritis according to the herbal medicine extract mixture treatment of the present invention. YNH431 is a herbal extract mixture; Normal is a negative control that did not induce inflammatory arthritis.
FIG. 11 shows the effect of the herbal medicine extract mixture of the present invention on the paw thickness and arthritis index of the model rats. YNH431 is a herbal extract mixture.
FIG. 12 is a graph showing the degree of suppression of the expression of inflammatory cytokines (IL-1β and TNF-α) in joint synovial tissues according to the herbal medicine extract mixture treatment of the present invention. YNH431 is a herbal extract mixture; β-actin is a loading control; Normal is a negative control that did not induce inflammatory arthritis.
FIG. 13 is a graph showing the effects of the herbal medicine extract mixture treatment of the present invention on inflammation and cartilage destruction of joints. YNH431 is a herbal extract mixture; Normal is a negative control that did not induce inflammatory arthritis.
FIG. 14 shows the results of inhibiting osteoclast formation in joints according to the herbal medicine extract mixture treatment of the present invention. YNH431 is a herbal extract mixture; Normal is a negative control that did not induce inflammatory arthritis.
Fig. 15 is a graph showing the effect of inhibiting joint destruction according to the herbal medicine extract mixture treatment of the present invention. (A) is a micro-CT photograph; (B) is a radiologic erosion score graph; YNH431 is a herbal extract mixture; Normal is a negative control that did not induce inflammatory arthritis.

In order to achieve the object of the present invention, the present invention provides a health functional food composition for preventing or ameliorating arthritis containing an extract of herbal medicine as an active ingredient.

The term "herbal medicine" as used in the present invention includes, but is not limited to, coriander, angelica, ovine, stigma,

In the health functional food composition for preventing or ameliorating arthritis of the present invention, the herbal medicine extract may be water, ethanol or an extract of a mixed solvent thereof, but is not limited thereto.

In addition, in the health functional food composition for preventing or improving arthritis of the present invention, the herbal medicine extract mixture may be prepared by 1) washing the stem, Angelica root, stem, contactal stem, rhizome root, Drying; 2) adding 0.8 to 1.2 L of ethanol and water to 200 g of each material dried in the step 1) and boiling at 70 to 80 ° C for 1 to 2 hours; 3) concentrating each of the extracts obtained in step 2) by filtration under reduced pressure, followed by lyophilization to obtain a powder; And 4) 30 to 50% by weight of the stem powder, 20 to 40% by weight of the angelica root powder, 5 to 15% by weight of the stem stem powder and 5 to 15% by weight of the talc stem powder obtained in the step 3) , 1 to 10% by weight of root powder, and 1 to 10% by weight of roots powder in the germination line, but the present invention is not limited thereto.

In the health functional food composition for preventing or improving arthritis of the present invention, the arthritis may be osteoarthritis or rheumatoid arthritis, preferably rheumatoid arthritis, but is not limited thereto.

The health functional food composition for preventing or ameliorating arthritis according to the present invention is characterized by the activity of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (ERK), or nuclear factor kappa B (NF-kB) But is not limited thereto.

The health functional food composition for preventing or ameliorating arthritis of the present invention may be manufactured by any one of powder, granule, ring, tablet, capsule, candy, syrup and beverage, but is not limited thereto.

The herbal medicine extract mixture may contain any one of the extract obtained by the extraction treatment, the diluted or concentrated liquid of the extract, the dried product obtained by drying the extracted liquid, the adjusted product, or the purified product.

The health functional food composition is not particularly limited as long as it can be ingested to prevent or ameliorate arthritis.

When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is, or may be used together with other food or food ingredients, and suitably used according to a conventional method. The active ingredient may be suitably used depending on its intended use (prevention or improvement). Generally, in the production of food or beverage, it is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the health functional food composition of the present invention. However, in the case of long-term intake for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the foods to which the health functional food composition can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, soups, Drinks, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

In addition, the health functional food composition of the present invention can be produced as a food, particularly a functional food. The functional food of the present invention includes components that are ordinarily added in food production, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, in the case of a drink, a natural carbohydrate or a flavoring agent may be included as an additional ingredient in addition to the active ingredient. The natural carbohydrate may be selected from the group consisting of monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, For example, xylitol, sorbitol, erythritol, etc.). The flavoring agent may be a natural flavoring agent (e.g., tau Martin, stevia extract, etc.) and a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).

In addition to the above-mentioned health functional food composition, it is possible to use various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, A carbonating agent used in beverages, and the like. Although the ratio of the added components is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition of the present invention.

The present invention also relates to a method for producing a plant extract, which comprises 30 to 50% by weight of a stem extract, 20 to 40% by weight of angelica root extract, 5 to 15% by weight of a stem extract, 5 to 15% % Root extract of Angelica keiskei koidz. And a mixture of 1 to 10 wt.% Of root extract of roast gland root as an active ingredient.

The pharmaceutical composition for improving or treating arthritis of the present invention may comprise 0.02 to 80% by weight, preferably 0.02 to 50% by weight, of the herbal extract mixture based on the total weight of the pharmaceutical composition.

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

The pharmaceutical dosage forms of the compositions according to the invention may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set.

The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method . Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition containing the herbal medicine extract mixture include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium A variety of compounds or mixtures including phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, . 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 or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like have. Formulations for parenteral administration include 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 the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for the desired effect, the herbal extract mixture of the present invention is preferably administered at 0.0001 to 100 mg / kg per day, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way. In addition, the pharmaceutical composition according to the present invention may be administered alone, or may be administered together with other medicines in the same or in order to supplement other medicines. In addition, in the composition of each formulation, components other than the composition, which is the above-mentioned essential ingredient, can be mixed and selected by a person skilled in the art according to the formulation or purpose of use of the other external preparation. In this case, Can happen.

The herbal extract mixture of the present invention can be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

Example  1. Herbal Extract Mixture ( YNH431 )

The roots, angelic roots, angelica roots, rhizomatous roots, stalk roots and roots of the godi line used in the present invention were purchased from Kyungdong market (Dongdaemun-gu, Seoul). The materials were washed, cut with a pulverizer, dried, and 200 g of the respective raw materials, ethanol and water were added to the extractor in an amount of 1 L each, and the mixture was extracted with hot water at 70 to 80 ° C for 1 to 2 hours. Only the extracted supernatant was separated, purified, and concentrated under reduced pressure using a rotary vacuum evaporator. Then, freeze-drying was carried out to obtain 7.2 g (yield: 3.6%), 49.52 g (yield: 24.76%) of angelic root, 12.22 g (yield: 6.11%) of clove, 27.84 g (yield: 13.92% g (yield: 4.67%) and 25.82 g (g, yield: 12.91%) of roots of mandarin root were obtained. The obtained powder was mixed with 30 to 50% by weight of stem, 20 to 40% by weight of angelica root, 5 to 15% by weight of ovine stem, 5 to 15% by weight of grafted stem, 1 to 10% Roots and 1 to 10% by weight of germination roots were prepared and used as the samples of the following examples.

Example  2. Synovial fibroblast  Herbal extract mixture for proliferation ( YNH431 )

Inflammatory arthritis is characterized by an inflammatory reaction caused by the hyperplasia of synovial fibroblasts, and stimulation of synovial fibroblasts with IL-1β induces hyperplasia of synovial fibroblasts. Therefore, in Example 2, the proliferation of synovial fibroblasts stimulated by IL-1β (interleukin-1β, 1.0 ng / mL) stimulation and the proliferation inhibitory effect of synovial fibroblast by the herbal extract mixture (100 μg / For synovial fibroblast, synovial fibroblasts were isolated from synovial tissues obtained during arthroscopic surgery of patients with rheumatoid arthritis. Separated synovial fibroblasts were cultured in 96-well U-bottom plates containing DMEM / FCS (Dulbecco's modified eagle's medium containing 10% FCS) at 1 × 10 ⁵ synovial fibroblasts per well and stimulated for 24 hours The cell proliferation was measured using a cell counting kit-8 (CCK-8 kit, Dojindo Laboratories, Japan). As a result, as shown in Fig. 1, the herbal extract mixture (YNH431) significantly inhibited the proliferation of synovial fibroblasts in a concentration-dependent manner at concentrations of 50, 100 and 200 占 퐂 / m, .

In inflammatory arthritis, the inflammatory response of synovial fibroblast hyperplasia is mediated through activation of various intracellular signaling molecules. In particular, mitogen activated protein kinases (MAPKs) and nuclear factor kappa B (NF-kB) play an important role in synovial fibroblasts. Therefore, Western blotting was carried out on MAPKs and NF-kB, which are signal transducing materials, in order to confirm the inflammatory response of the herbal medicine extract mixture (YNH431) and the proliferation inhibitory effect of synovial fibroblasts. (EACT) and Angelica gigas Nakai extract (EAAS), which are reported to have inhibitory activity against the inflammatory reaction and synovial fibroblast proliferation, Inhibitory activity was increased. As a result, it was confirmed that the herbal medicine extract mixture (YNH431) inhibited the expression of ERK, p38 MAPKs and NF-kB by IL-1β as shown in FIG. 2, thereby statistically inhibiting the hyperplasia of synovial fibroblast there was. In addition, inhibition of inflammatory reaction and synovial fibroblast proliferation in the treatment of herbal medicine extract (YNH431) was more inhibited than that of herbal medicine extracts reported to have inhibitory activity against inflammatory reaction and synovial fibroblast proliferation such as extracts of CJ, , The herbal medicine extract mixture (YNH431) of the present invention may be more effectively used for preventing, improving or treating arthritis.

Example  3. Herbal extract mixture ( YNH431 ) And each herbal medicine extract In synovial fibroblasts  Inflammatory response inhibitory effect

IL-1β (1.0 ng / mL), which is important for causing arthritis, was treated with IL-1β (1.0 ng / mL) for 24 hours to induce inflammatory reaction, and inflammatory reaction and joints were observed in synovial fibroblasts treated with each herbal extract and herbal medicine extract (YNH431) On the production of MMPs, TIMPs, COX-2, and PGE2, which cause destruction of the cells. Based on the results of Example 2, the expression levels of MMPs, TIMPs, COX-2 and PGE2 in the herbal extract mixture (YNH431) and herbal medicine extracts were analyzed. In addition, the extracts of the extracts of Pan, Cipiporan and Angelica gigantum, which are reported to have an inhibitory activity against the inflammatory reaction in synovial fibroblasts, or the herbal extract mixture (YNH431), which is an effective ingredient of the present invention, The expression levels of proteins related to inflammation were compared with those of herbal extract mixtures and 4 kinds of herbal extract mixtures.

As a result, the expression of COX-2, MMP-1, 2, 3 and PGE2 by IL-1β was inhibited when each crude herbal extract and herbal medicine extract mixture (YNH431) were treated as shown in FIGS. 3 and 4, Lt; RTI ID = 0.0 > TIMP-1 < / RTI > In addition, as compared with the case of extracting the herbal extracts, extracts of cucurbitan and angelica guinea, or extracts of herb extracts of the three kinds (extracts of rape, cucumber and angelica) and herb extracts of four kinds (herb, cucumber, The expression of COX-2, MMP-1, 2, 3 and PGE2 by IL-1β was significantly decreased in the case of treatment with 6 herbicidal extracts (YNH431) As shown in Fig.

These results indicate that IL-1β inhibits synovial fibroblast proliferation, MMPs, COX-2 and PGE2 expression through MAPKs and NF-kB pathway, which are intracellular signaling substances, It is believed that the treatment of the herbal medicine extract mixture (YNH431) of the present invention has a better inhibitory effect on inflammation than the treatment of the four herbal medicine extract mixtures.

Example  4. Depending on the mixing ratio of herbal medicine extracts Of synovial fibroblasts  Inflammatory response inhibitory effect

The synovial fibroblasts were cultured in a 96-well plate containing DMEM / FCS (Dulbecco's modified eagle's medium containing 10% FCS) at a concentration of 1 × 10 ⁵ cells / well. IL-1β (1.0 ng / mL), an important inflammatory cytokine to cause arthritis, was treated for 24 hours to induce an inflammatory reaction and whether the herbal extract mixture (YNH431) inhibited the inflammatory response. The mixing ratio of the herbal extracts was 3: 2, 4: 2 and 4: 3, respectively, and the other medicinal herbs such as mandarin, guangdong, 1: 1: 0.5: 0.5. Therefore, in the above three ratios of herbal medicine extraction mixtures, MMPs (matrix metalloproteinases), Tissue inhibitor of matelloprteinase (TIMPs), which inhibit MMPs, and inflammatory responses of synovial fibroblasts, which play an important role in the pathogenesis of arthritis, The expression of COX-2 (cyclooxygenase-2) was analyzed by Western blotting. As a result, when the proportion of the herbal medicine extract mixture (YNH431) of the present invention was 4: 3: 1: 1: 0.5: 0.5 (per serving: COX-2, and MMP-1, 2, and 3 (FIG. 5).

Example  5. Herbal extract mixture ( YNH431 Inhibition of osteoclast differentiation

In inflammatory arthritis, the role of osteoclasts is very important because the inflammatory response destroys joints through differentiation and activation of osteoclast precursor cells to osteoclasts. Such a method of inhibiting the role of osteoclasts is becoming a treatment method of arthritis. Therefore, bone marrow cells were isolated from the bone marrow of the 5 - week - old imprinting control region (ICR) mouse femur and used as precursor cells of osteoclasts. 3 × 10 ⁴ osteoclast precursor cells were cultured in α-MEM (Alpha-Minimum Essential Medium) containing 10% FBS (fetal bovine serum, Gibco BRL) and antibiotics in a 10 cm culture plate. (30 ng / mL) and RANKL (Receptor activator of nuclear factor kappa-B ligand, 50 ng / mL) to induce differentiation into osteoclasts. To examine the effect of YNH431, cultures were stimulated for 3 days with various concentrations of YNH431 (10, 50, 100 and 200 ng / mL) and IL-1β (1 ng / mL). The osteoclasts were stained with TRAP (Tartrate-resistant acid phosphatase), which indicates osteoclast differentiation, and cells with more than 5 nuclei were stained. In addition, the calcium phosphate thin film- 3 × 10 ⁴ osteoclast precursor cells per well were cultured on well tissue culture plates (OAAS plates, Oscotec, Cheonan, Korea). After 2 weeks, image analysis system (Image Gauge, Tokyo, Japan) The area of resorption pits at randomly selected sites was calculated. As a result, as shown in Fig. 6, the crude herbal extract mixture showed significant osteoclast differentiation in a concentration-dependent manner at a concentration of 0, 10, 50, 100 and 200 / / mL in TRAP staining of osteoclast differentiation The inhibition of osteoclast differentiation by IL-1β was significantly inhibited by the herbal extract mixture treated at a concentration of 100 μg / mL. This effect was also confirmed by studies using resorption pits (Fig. 7).

In addition, activation of various signal transduction materials involved in osteoclast differentiation was measured to understand the mechanism of YNH431 inhibition of osteoclast differentiation and activation. (30 ng / mL), RANKL (50 ng / mL), and IL-1 (1.0 ng / mL) were isolated from bone marrow cells (3 x 10 ⁴ cells / well) from the bone marrow of a 5-week-old imprinting control region / mL) and herbal extract mixture (100 mg / mL), total RNA and protein were obtained and analyzed by RT-PCR and Western blotting. As a result, YNH431 inhibited the expression of NFATc1, c-Fos, TRAP, E-cadherin, cateptin K, c-Src, MAPKs and NF-kB by IL- .

Example  6. Herbal Extract Mixture in Arthritis Animal Model ( YNH431 ) Of arthritis

To prepare an inflammatory arthritis animal model (CIA, collagen-induced arthritis), 100 μg of umbilical Ⅱ collagen (subcutaneously injected) was injected subcutaneously into the tail of 7-10 week old Inbred male DBA / 1 mice (SLC Inc, JAPAN) type Ⅱ collagen, Chondrex Inc, USA) and FCA (Freund's complete adjuvant, Chondrex Inc, USA) were injected into the abdominal cavity with booster-injection on the 21st day.

To evaluate the effect of YNH431, group 3 was divided into 10 groups. Group 1 did not induce arthritis as a negative control. Group 2 received 100 μl of vehicle. Group 3 received 10 mg / kg YNH431 was administered after booster-injection, and the degree of arthritis and changes in joint tissue were observed on days 3, 18, and 28 (FIG. 9).

(1) Activity of arthritis and inflammatory cytokines IL -1β and TNF -α production inhibitory effect

In collagen-induced arthritis (CIA), an animal model of inflammatory arthritis, YNH431 inhibited the hind-paw inflammation compared to the vehicle-treated group, and the type II collagen was added to the booster-injection ), And the frequency of inflammatory arthritis was observed to be statistically significantly decreased when observed for 9 days (Fig. 10).

The inflammatory arthritis animal model, CIA, examined the severity of arthritis using paw thickness and arthritis index for 18 days after booster-injection of type II collagen. YNH431 significantly reduced the paw thickness and arthritis index in the CIA compared to the vehicle-treated group (control group), thus inhibiting the development of arthritis and lowering the activity of arthritis (Fig. 11).

In order to determine whether the inhibitory effect of inflammatory arthritis caused by the herbal medicine extract (YNH431) influences the expression of inflammatory cytokines, which play an important role in the pathogenesis of arthritis, booster- The expression levels of IL-1β and TNF-α were analyzed by Western blotting. The herbal extract mixture (YNH431) inhibited IL-1β and TNF-α, which are important cytokines in inflammation of joints in synovial tissues of knee and ankle joints compared to the vehicle-treated group in CIA (Fig. 12). As shown in Fig.

(2) Analysis of inhibition of arthritis in histologic findings

In the collagen-induced arthritis (CIA), an animal model of inflammatory arthritis, type II collagen was added to confirm the gross effect of YNH431 in the joint tissue, as compared to the vehicle-treated group, At 28 days after booster-injection, knee and ankle joint tissues were obtained. To determine the inflammatory response, hematoxylin and eosin staining (100X), trapezinin-0 staining (100X), and TRAP staining (200X ). As a result, as shown in FIG. 13, YNH431 was found to be superior to knee and ankle joints in the control group, CIA, as a result of hematoxylin and eosin (100 & Inflammation such as infiltration of tissue inflammatory cells was suppressed (Fig. 13, left). The results of the saprinin-0 staining (100X) showed that the CIA inhibited articular cartilage destruction of the knee and ankle joint tissues in comparison with the control vehicle group (Fig. 13, right). In addition, the CIA results of TRAP staining (200X) showed that osteoclast infiltration, which causes destruction of knee and ankle joint tissue, is suppressed and joint destruction is reduced compared with the control group, (Fig. 14).

(3) Micro- CT Of arthritis inhibition

In order to investigate the effect of YNH431 on collagen-induced arthritis (CIA), which is an animal model of inflammatory arthritis, on the degree of joint destruction compared to vehicle-treated control group, type II collagen was booster- (Skyscan 11.70, Kontich, Belgium; X-ray voltage: 60 kV; current: 100 μA; 1,000 × 600 large camera resolution) were sacrificed on the 28th day after sacrifice. As a result, as shown in FIG. 15, YNH431 significantly inhibited the destruction of joints in the micro-CT test of the knee, ankle and paw in the CIA compared to the vehicle-treated group in the CIA And radiologic erosion score analysis showed that YNH431 significantly suppressed joint destruction by reducing erosion.

Claims (10)

A root extract of 20 to 40 wt%, a root extract of Angelica keiskei koidz., A root extract of 5 to 15 wt%, a root extract of 5 to 15 wt%, a root extract of 1 to 10 wt% And 1 to 10% by weight of a roots extract of rosemary root as an active ingredient. delete delete The method of claim 1,
1) Washing the stem, the root of Angelica gigas, the stem of the stem, the stem of the stigma, the root of the stem,
2) adding 0.8 to 1.2 L of ethanol and water to 200 g of each material dried in the step 1) and boiling at 70 to 80 ° C for 1 to 2 hours;
3) concentrating each of the extracts obtained in step 2) by filtration under reduced pressure, followed by lyophilization to obtain a powder; And
4) a mixture of 30 to 50% by weight of the stem powder, 20 to 40% by weight of the angelica root powder, 5 to 15% by weight of the stem stem powder, 5 to 15% 1 to 10% by weight of root powder and 1 to 10% by weight of roots powder of a medicinal herb. The health functional food composition according to claim 1, wherein the arthritis is osteoarthritis or rheumatoid arthritis. The composition of claim 1, wherein the composition is selected from the group consisting of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), or NF-kB (nuclear factor kappa-light chain enhancer of activated B cells) Wherein the activity is inhibited. The health functional food composition according to claim 1, wherein the composition is prepared from one of powder, granule, ring, tablet, capsule, candy, syrup and beverage. A root extract of 20 to 40 wt%, a root extract of Angelica keiskei koidz., A root extract of 5 to 15 wt%, a root extract of 5 to 15 wt%, a root extract of 1 to 10 wt% And 1 to 10% by weight of a germline root extract as an active ingredient. The pharmaceutical composition according to claim 8, further comprising a pharmaceutically acceptable carrier, excipient or diluent in addition to the mixture. [Claim 9] The pharmaceutical composition according to claim 8, wherein the composition is prepared by any one of a capsule, a powder, a granule, a tablet, a suspension, an emulsion, a syrup and an aerosol.

Images