KR101248378B1 - Pharmaceutical composition for arthritis treatment and prevention - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing a complex crude drug extract is provided to suppress synthesis of a substrate disruptor and to maximize a cartilage protection function. CONSTITUTION: A pharmaceutical composition for preventing and treating arthritis contains Caragana sinica, Gentianae macriphyllae Radix, Kalopanacis Cortex, Eucommia ulmoides, Ledebouriellae Radix, Clematidis Radix, Achyranthes japonica (Miq.) Nakai, and Acorus gramineus in a weight ratio of 1:0.5-3:0.05-1:0.5-3:0.5-2.5:0.5-3:0.5-3:0.5-3. [Reference numerals] (AA) Complex crude drug extract; (BB) Negative control group; (CC) Positive control group; (DD) Comparative example 1; (EE) Comparative example 2; (FF) Comparative example 3; (GG) Preparation example 1;

Description

Pharmaceutical composition for Arthritis treatment and prevention

The present invention relates to a pharmaceutical composition for treating and preventing arthritis.

Arthritis is a representative degenerative and refractory disease that affects more than 2 million people in Korea, where 5% of the world's population suffers. It is a disease that causes swelling and pain in the joints of the whole body due to inflammatory changes in the synovial membrane of the inner capsule capsule. . Arthritis is a progressive and difficult joint deformity and articulation that causes severe discomfort if not treated. Arthritis is largely classified into degenerative arthritis (osteoarthritis) and rheumatoid arthritis.

The degenerative arthritis is a disease that occurs mainly in middle age or old age and causes pain, stiffness, and movement disorders in weight-bearing joints such as knee joints and hip joints. As a cause, I thought that aging is a aging phenomenon caused by age, but now it is known that there are many causes. Symptoms are thought to be caused by a combination of age, genetic factors, obesity, joint shape, and hormones. Degenerative arthritis is primarily involved in cartilage damage, and matrix metalloproteinases (MMPs), known to be involved in cartilage damage, destroy cartilage tissue. Causes degenerative changes. Induced inflammatory cytokines, interkukin-1 (IL-1), and tumor necrosis factor-a (TNF-a), which also affects tissues such as muscles and ligaments, causing severe pain. (Fernandes JC et. Al., The role of cytokines in osteoarthritis pathophysiology, 39, pp237-46, 2002).

In addition, the rheumatoid arthritis is an inflammatory autoimmune disease in various joints. In patients with arthritis, hyperplasia of the synovial membrane tissue of the joints occurs, while macrophages, dendritic cells, and activated T lymphocytes and B lymphocytes migrate to the synovial tissue and polymorphic cells Polymorphonuclear cells accumulate in synovial fluid and on cartilage surfaces, causing inflammation. Inflammation of the synovial tissue is thought to be caused by T lymphocytes reactive to unknown autoantigens.

Nevertheless, T lymphocytes that infiltrate most tissues show no activation markers on the cell surface and rarely express cytokines. In contrast, macrophage-derived cytokines have been observed in synovial tissues and blood with rheumatoid arthritis symptoms. Such cytokines include interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-a), which are known to promote the growth of synovial fibroblasts. These experimental results suggest that T lymphocytes are very important for inducing inflammation of synovial tissue and that subsequent inflammatory symptoms are maintained by cytokines derived from activated synovial cells (carson DA et. Al., J Clin.Invest., 87, pp 379-383, 1991 ,; panayi GS, curr.opin.Rheumatol., 9, pp236-240, 1997).

In addition, synovial hyperplasia can form irreversible destruction of cartilage and bone tissue of diseased joints by forming pannus, a layer of connective tissue that develops in cartilage without blood vessels in arthritis. Metalloproteinases (MMPs) and the like act to destroy cartilage tissue.

The ideal goal of all arthritis, such as degenerative arthritis and rheumatoid arthritis, is to protect the cartilage and bone tissue of the diseased joint. It has been reported that activating IL-4 (Interleukin-4), a cytokine involved in maintaining cartilage, has an effect on cartilage protection (Kim SH, J. Immunol., 166, pp3499-3055, 2001). .

In addition, MMPs are well known as cartilage destruction factors, and TIMP (tissue inhibitor of metalloproteinase) is an inhibitor of this enzyme. Among the MMPs, MMP-2 exhibits physiological activity that is important for normal remodeling of connective tissues, but induces severe tissue damage in arthritic joints. Therefore, MMP-2 is inhibited and an inhibitor of this enzyme. It has been reported that the development of drugs with inducible activity of TIMP-2 not only prevents cartilage destruction, which is one of the most important symptoms of arthritis, but also prevents severe pain of patients (Mengshol JA et.al). , Arthritis Rheum., 46, pp 13-20, 2002). No such arthritis treatments have been developed yet, and the drugs used so far have very limited effects, causing severe pain in patients once arthritis occurs.

Therefore, the purpose of treating arthritis is to reduce the pain and inflammation of the joint, to prevent the deformation of the joint, and more fundamentally to identify and control the mechanism of intracellular action that causes arthritis.

Meanwhile, known arthritis therapeutic agents include suitable nonsteroidal anti-inflammatory drugs (NSAIDs), contraindications, penicillamine, penicillamine, and steroid hormones. However, such a therapeutic agent has a problem in that side effects are taken for a long time. Recently, efforts have been made to produce a water-soluble receptor of TNF (tumor necrosis factor), which plays a central role in inflammatory mechanisms, as a therapeutic agent.However, inflammation, edema, abnormal neovascularization, erosion of bone and cartilage tissue, etc. Unexpected side effects occur. Therefore, there is an urgent need to develop a new therapeutic agent having relatively low side effects when applied in vivo and excellent in alleviating the symptoms of arthritis.

 Fernandes J.C. et. al., The role of cytokines in osteoarthritis pathophysiology, 39, pp 237-46, 2002  Mengshol J.A. et.al., Arthritis Rheum., 46, pp 13-20, 2002

In order to maximize the therapeutic effect, especially cartilage protection effect against arthritis, the present inventors to maximize the characteristics and efficacy of each of these herbal medicines from the complex herbal medicines to promote the function of cheonghap, gangeun bones, blood circulation and tissue regeneration Based on the selection of about 100 candidate herbal medicines based on this, through the experimental experiments for the treatment of arthritis to find a combination of herbal drugs having a remarkable arthritis treatment effect was completed the present invention.

It is an object of the present invention to provide a pharmaceutical composition for the treatment and prevention of arthritis, containing a complex herbal extract as an active ingredient.

In addition, it is an object of the present invention to provide a health functional food for preventing and improving arthritis containing a complex herbal extract as an active ingredient.

The present invention provides a composite herbal extract that can inhibit the synthesis of glass and substrate destruction of inflammatory substances to maximize the cartilage protection effect, to achieve the above object, and to treat and prevent arthritis as an active ingredient To provide.

In addition, the present invention provides a functional food for preventing and improving arthritis containing the complex herbal extract as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the treatment and prevention of arthritis, containing a complex herbal extracts including goldamcho, Jinkyo, Haedongpi, tongchung, windproof, gastric, hyssop, and Seokchangpo as an active ingredient.

Caragana Chambgu Lamarck of the present invention is 1 to 2 cm tall and leafy bush. The leaf is an even pinnip leaf consisting of two pairs of oval-shaped leaves, attached to long branches and gathered on short branches. There is a thorn under the leaf stem. Branches with leaves are used, with flavonoids on the leaves and branches and alkaloids on the bark. It is prescribed as an effective medicine for gastrointestinal diseases, myalgia, and neuralgia.

Gentianae Macrophyllae Radix of the present invention is dried root of Gentiana macrophylla pall and gentian. The stem reaches 2 m in height and leaves are rounded in 3 to 5 branches. Blue flowers bloom in summer. At the beginning there are flavonoids, 0.8% in leaves, 0.69% in stems, 0.25% in flowers, and 1.3-1.7% alkaloids in roots. Roots are known to have been used for lowering fever, rheumatism, arthritis, and limb paralysis in the treatment of sinus (biopharmaceuticals, ming ming, pp165).

The thawing skin (Kalopanacis cortex ) of the present invention is kalopanax pictus nakai , stems of the Ogalpi tree). The outer part is dark brown and the inside is fennel color with many long spines. All parts of the plant have saponies, rectifications and coumarins. Flavonoids are pronounced in flowers and slightly reactive in stems and leaves. It is known that copper therapy has been used for anti-arthritis by suppressing analgesics and inflammatory reactions (Biopharmaceuticals, Dongmyung, pp549).

Eucommiae of the present invention Cortex ) is a bark of the family of the cedar , which is mainly produced in China, and is also cultivated in Korea and Japan. It is a leafy tree, and there is rubber throughout the plant, so when the bark and leaves boil, silk-like lines are formed. The leaves contain chloric acid (C ₁₆ H ₁₈ O ₉ ) caffeic acid, acuvin (C ₁₅ H ₂₂ O ₉ ) gum. In motion therapy, it is known to be used for blood pressure strengthening, low back pain and knee joint, and especially to promote collagen synthesis (Li Y, et. Al., (2000) Biol. Pharm. Bull. 23; 54 ~ 59).

Windproof of the present invention ( Saposhnikoviae radix ) is circumferential, 15-20 cm long, 7-15 mm in diameter, with a slightly thin rhizome on the lower part. At the root is essential oil and 0.23 to 0.88% coumarin. Since ancient times, it has been known to have various wind and head pain, bone node pain and arthritis inhibitory effects (biopharmaceutical, gyeongmyeongsa, pp137).

Of the present invention wiryeongseon (clematidis radix ) It grows about 4m long and native to China, and leaves are viviparous and run 3 times or sometimes 3 times. Leaflets are ovate or ovate lanceolate, 2-5 cm long, without hairs on the surface but with fine hairs on the back and sparsely on the edges, and the roots and rhizomes of the same family. Root contains saponin and about 0.2% flavonoids, separated from querchitin and camme. In the treatment of motion, it is known that wind and humidity have been used for muscle paralysis, joint paralysis, rheumatism, and gouty arthritis.

Achyranthis radix of the present invention is a perennial herb of about 90 cm in height, the stems are square, and the nodes are swollen like a knee and are roots of iron knee or amaranth. The roots contain saponins that break down into oleanolic acid and glucuronic acid when hydrolyzed. Alkaloids, water-soluble akyratin (C ₆ H ₁₁ O ₂ NH ₂ O) and other alkaloids and ash, 8% of which is 24.5%. It is known that it has been used for anti-inflammatory action, joint pain improvement, stroke, and menstrual irregularities.

Acori graminei rhizoma of the present invention is a root stem of araceae, which is a long blade-shaped leaves gathered by perennial grasses grown in the water. There are 0.5 to 0.9% of essential oils in the rhizome, and 63 to 81% of essential oils have β-asarone. In addition, there are palmitic acid and phenolic trace elements. It is known that the treatment of motion is effective for health, sedation, and antifungal activity (Biopharmaceuticals, Dong Myung Co., pp253).

The composition of the present invention is characterized in that the complex herbal extract containing goldamcho, jinkyo, Haedongpi, Tofu, Windproof, Maeseryeong, Woo Seol, and Seokchangpo as an active ingredient.

In the present invention, the 'composite herbal medicine extract' means an active ingredient separated from the herbal medicine, that is, a mixture of substances showing a desired activity, and is usually prepared by an extraction process using water, an organic solvent or a mixed solvent thereof, Extracts of organic solvents or mixed solvents thereof, dry powders thereof, or any form formulated using them.

In addition, the complex herbal extracts include those fractions obtained by the extraction process.

The complex herbal extract may be prepared using water, alcohol, or a mixed solvent thereof as an extractant, and preferably, may be extracted with a mixed solution of water and alcohol.

In particular, the solvent may be a mixed solvent of water and an alcohol having 1 to 4 carbon atoms, more particularly, an alcohol having 1 to 4 carbon atoms such as methanol and ethanol of 3 to 30%, and still more preferably 5 to 20% ethanol. have.

The composite herbal extract of the present invention may be prepared by separately extracting each herbal medicine as an extract, or may be extracted by mixing the herbal medicine.

Complex herbal extract of the present invention can be prepared in more detail as follows.

The complex herbal extract is dried after the combined herbal medicine of goldamcho, Jinkyo, Haedongpi, Toe Chung, Windbreak, Ulleung, Woo Seol, and Seokchangpo, and then added water, lower alcohol or a mixed solvent of 5 to 20 times the volume of the combined herbs to 80 Extracting soluble in a solvent by repeatedly extracting 2 to 5 times by hot water extraction for 1 to 24 hours at an extraction temperature of 100 ° C .; And

After filtering the extract obtained, and then concentrated under reduced pressure at 40 to 80 ℃ to prepare a powder extract through lyophilization or vacuum drying; may be prepared.

In addition, the available extract can be further subjected to the fractionation process with a solvent selected from the group consisting of hexane, methylene chloride, acetone, ethyl acetate, chloroform or a mixture thereof. In the fractionation, two or more solvents may be used individually or in combination, and may be used sequentially according to the polarity of the solvent to prepare extracts of each solvent. Extract preparation temperature may be 4 to 120 ℃, but is not limited thereto. The extraction time is not particularly limited, but may be 10 minutes to 30 days, and a conventional extraction device, an ultrasonic grinding extractor, or a fractionator may be used. The prepared extract can then be filtered under reduced pressure or / and lyophilized to remove the solvent.

The compound herbal extract of the present invention has a compounding weight ratio of 1, 0.5 to 3: 0.05 to 1: 0.5 to 3: 0.5 to 2.5: 0.5 to 3: 0.5 to 3: 0.5 to 3.

Complex herbal extracts, when used in certain combination weight ratios of the gallweed, jinkyo, Haedongpi, tonggi, windbreak, gastrointestinal, hyssop, and Seokchangpo, compared to when treated with some combinations of the herbal medicine, tumor necrosis factor-alpha (TNF- The inhibitory effect of inflammatory substances such as α) and nitric oxide (NO) is about 4 to 5 times better and the synthesis of matrix metalloproteinase-13, which is a substance that destroys cartilage, When treated at the same temperature, it can be effectively used for the prevention or treatment of the desired arthritis by significantly inhibiting.

The compounding weight ratio of goldamcho, Jinkyo, Haedongpi, Toesok, Windbreak, Gasyeongseon, Wooslung, and Seokchangpo have important meanings on the effectiveness of treating the arthritis and the practical effect of the material showing the desired activity in the herbal medicine. There is an efficient and economical advantage by appropriately limiting the blending ratio of each extract.

In particular, the compounding weight ratio of Goldamcho, Jinkyo, Haedongpi, Toesok, Windbreak, Gasyeongseon, Wooslung, and Seokchangpo is from 1: 1 to 2: 0.1 to 0.5: 1 to 2: 0.7 to 1.5: 1 to 2: 0.7 to 1.5: 0.7 to 0.7. In the range of 2, it shows a significant synergistic effect in the treatment of arthritis and can be used as an effective arthritis treatment in place of the previous treatment.

When extracts containing gallweed, orthodontic, thawing blood, worms, windbreaks, gastrointestinal tract, hyssop, and Seokchangpo are used in complex compositions, matrix inhibitors (MMPs) that inhibit free radicals and cartilage of inflammatory substances, one of the main causes of arthritis, -13) was effectively inhibited, and the progression of arthritis was also effectively suppressed for chronic arthritis.

This proves that the composition of the present invention has an excellent effect as a treatment for arthritis.

The composition of the present invention may be used in an active ingredient of 0.001 to 95% by weight, preferably 1 to 80% by weight of the total 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.

Therefore, the complex herbal extract of the present invention shows excellent activity in inhibiting the synthesis of tumor necrosis factor-alpha and nitoxide and matrix metalloprotease-13, which are inflammatory substances, and inhibiting the synthesis of arthritis in arthritis animals. Since it has the effect of significantly inhibiting pathological progression, it can be effectively used for the prevention or treatment of arthritis.

The arthritis includes acute and chronic rheumatism; Atrophic arthritis; Chronic inflammatory arthritis; Collagen-induced arthritis; Deformable arthritis; Infectious arthritis; Menopausal arthritis; Break arthritis; Hypertrophic arthritis; Purulent arthritis; And arthritis and degenerative osteoarthritis diseases caused by tuberculous arthritis; and preferably, may be acute rheumatoid, chronic rheumatism, collagen-induced arthritis and chronic inflammatory arthritis.

The present invention is a combination herbal extract containing the above-mentioned goldamcho, Jinkyo, Haedongpi, Tohpi, windbreak, gastric, hyssop, and Seokchangpo in combination with foods, for example, health supplements, functional foods, food additives, etc. Can be. The health supplements, functional foods, food additives have an action for the treatment or prevention of arthritis.

As used herein, the term "prevention" refers to any action that inhibits or delays the development of arthritis by the administration of a composition comprising a complex herbal extract comprising boneweed, orthodontist, thawing blood, worms, windbreak, gastrointestinal tract, hyssop, and Seokchangpo. it means.

As used herein, the term "treatment" refers to any or all of which improves or beneficially alters the symptoms of arthritis by administration of a composition comprising a complex herbal extract comprising bonsai, necrosis, thawing blood, worms, windbreaks, gastrointestinal tract, hyssop, and Seokchangpo. It means an act.

Pharmaceutical compositions comprising the compositions according to the invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. Pharmaceutically acceptable carriers, excipients and diluents that may be included in the pharmaceutical composition according to one embodiment of the invention include lactose, dextrose, glucomannan, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia Rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate And mineral oils.

The pharmaceutical composition according to the present invention can be formulated and used in various formulations such as tablets, capsules, granules, pills, suspensions, syrups and the like according to conventional methods known in the art.

The pharmaceutical composition comprising a complex herbal extract comprising boneweed, pendulum, thawing blood, worms, windbreaks, gastric glands, hyssop, and Seokchangpo of the present invention not only provides excellent arthritis treatment or preventive effect, but also toxicity and side effects by drugs. It can be used with confidence even during long-term use. Accordingly, the complex herbal extract including goldamcho, Jingyo, Haedongpi, Toe Chung, Windproof, Gasyeongseon, Woo Seol, and Seokchangpo can be used as a main, secondary ingredient and food additive of food.

Therefore, in this respect, the present invention is a combination herbal extract containing the above-mentioned goldamcho, Jinkyo, Haedongpi, tofu, windproof, gastric, hyssop, and Seokchangpo in combination with food, for example, dietary supplement, functional food, It may be provided as a food additive. The dietary supplement, functional food, food additives have an action for the treatment or prevention of arthritis.

As used herein, the term "food" means a natural product or processed product containing one or more nutrients, and preferably means that it can be directly eaten through a certain processing process, By the meaning, it is intended to include all foods, food additives, functional foods and beverages.

As used herein, the term "functional food" refers to the regulation of biodefense rhythms of food groups or food compositions that have added value to the food by using physical, biochemical, or biotechnological techniques to act and express the function of the food for a specific purpose. , Means a food processed and designed to fully express the bioregulatory function related to disease prevention and recovery to the living body, and particularly includes "health functional food".

As used herein, the term "health food" is a food manufactured and processed using raw materials or ingredients having a useful function for the human body, and is useful for health uses such as nutrient control or physiological action on the structure and function of the human body. Refers to foods that are manufactured and processed by extracting, concentrating, refining, or mixing certain ingredients as raw materials or ingredients contained in food raw materials for the purpose of health supplement to obtain effect.

In particular, the functional food or health supplement in the present invention means a food that can improve the symptoms of arthritis or help to reduce the progression of arthritis, compared to before ingesting the food, and the general knowledge in the art If you have a medical history, you can know the exact criteria of arthritis and judge the degree of improvement by referring to the data presented by the Korean Medical Association.

The functional foods and dietary supplements may further include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of functional foods.

Examples of foods to which the complex herbal extracts including goldamcho, jinkyo, Haedongpi, tofu, windbreak, gastric, hyssop, and Seokchangpo of the present invention can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, and functionalities. Food and the like. In addition, the food in the present invention includes special nutritional products (e.g., prepared oils, infants, baby food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), health supplements, seasoned foods ( For example, soy sauce, miso, red pepper paste, mixed soy sauce, sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.), beverages ( Examples include, but are not limited to, fruits, vegetable drinks, soy milk, fermented beverages, and the like, and natural seasonings (eg, ramen soup). The food, beverage or food additive may be prepared by a conventional production method.

Functional drink in the present invention is a generic term for drinking to quench thirst or to enjoy the taste and is intended to include a functional drink. The beverage is not particularly limited to other ingredients other than including the complex herbal extracts including gallweed, Jingon, Haedongpi, Tofu, Windproof, Gasyeongsen, Emulsion, and Seokchangpo as essential ingredients in the indicated ratios, and there is no special limitation. Various flavors or natural carbohydrates, etc. may be included as additional ingredients. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose and other disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, and Sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1 to 20 g, preferably 5 to 12 g per 100 ml of the composition of the invention, else the composition of the invention is intended for the preparation of natural fruit juices, fruit juice drinks, vegetable drinks. It may further contain a pulp for.

Each extract constituting the composition of the present invention is a natural food with little toxicity and side effects, so can be used with confidence even for long-term use for the purpose of prevention.

The pharmaceutical composition according to the present invention is mixed with each herbal extract in a specific ratio capable of exerting the free inhibitory ability of an inflammatory substance which is one of the main causes of arthritis, and thus there is no significant synergy as well as side effects in the treatment of arthritis. There is an advantage that can be used as an effective arthritis treatment by replacing the treatment of.

1 is a view showing the effect of the composite herbal extract of the present invention on the inhibition of the synthesis of matrix metalloproteinase-13 (MMP-13), which is a cartilage matrix destruction substance.

The invention will be described in more detail with reference to the following examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.

Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

[Production Example 1]

Goldamcho, Jingyo, Haedongpi, Doochung, Windproof, Weeyeongseon, Wooslung, Seokchangpo were produced and used in commercial building. Each herb was washed thoroughly and dried in the shade.

Goldamcho (25.2g), Jingyo (37.8g), Haedongpi (7.5g), Tofu (42g), Windbreak (28.5g), Maeseryeongseon (36g), Wooseul (27g), Seokchangpo (30g) was prepared by crushing finely. .

4 L of 10% ethanol mixed solvent was added to the composite herbal medicine, the mixture was stirred well, and heated to reflux for 3 hours at an extraction temperature of 90 to 95 ° C., and then the filtrate was filtered through black paper. The filtrate was collected over and concentrated under reduced pressure the herbal extract at 60 ℃ and then lyophilized to obtain 27 g of the mixed herbal composition powder extract (DH301-2).

Comparative Preparation Example 1

A powder ax was prepared in the same manner except adding 4 L of distilled water to the ground herbal medicine of Preparation Example 1.

[Comparative Production Example 2]

Powder ax was prepared in the same manner except that 4 L of 50% ethanol mixed solvent was added to the pulverized herbal medicine of Preparation Example 1.

Example 1

In the production methods of Preparation Examples and Comparative Preparation Examples 1 and 2, the yield of the composite herbal medicines according to the extraction method was investigated. That is, the extraction according to the solvent was performed three times each, and the yield according to the number of extraction was expressed as a percentage of the total yield.

As can be seen in Table 1, the hot water extraction method using the alcohol mixed solvent of Example 1 was found to be extracted almost close to 100% only by the second extraction compared to the extraction method of the comparative example.

The result is a result confirming that there is a difference in the pharmacological efficacy of the active ingredient contained in the complex herbal medicine as well as the economic problem of the extraction method, the effect according to the treatment for a certain period of time.

[ Experimental Example  1] Tumor Necrosis Factor-a by Complex Herbal Extracts TNF -a) free inhibition efficacy assay

In order to investigate the effect of the composite herbal extract of Preparation Example 1 on the free inhibition of the tumor necrosis factor-a (TNF-a), a cytokine released from THP-1 cells, which are human monocytic cell lines The following experiment was carried out.

In order to compare the excellent efficacy of the composite herbal extract of Preparation Example 1, it was prepared in the same manner as the extraction method of Preparation Example 1, but was used in the experiment by varying the combination of the composition of the composite herbal medicine.

That is, in Comparative Example 1, a composite herbal extract prepared by mixing goldamcho, Tobacco, Jinkyo, Garyeongseon, Seokchangpo so that the blending weight ratio is 1: 1.7: 1.5: 1.4: 1.2.

In addition, as a comparative example 2, a composite herbal extract prepared by mixing gallweed, tofu, Seokpyo, windproof, dew, gallweed and thawed blood so that the blending weight ratio is 1: 1.7: 1.2: 1.1: 1.0.

In addition, in the Comparative Example 3, a composite herbal extract prepared by mixing goldamcho, Jinkyo, Gasyeongseon, windbreak, and dew with a blending weight ratio of 1: 1.5: 1.4: 1.1: 1.0.

(1) Cell selection and cell culture

In order to evaluate the effect of the composite herbal extract of Preparation Example 1 on the release of human tumor necrosis factor-a (TNF-a), THP-1 cells (ATCC No.TIB-202), a human-derived cell line, Purchased from Cell Line Bank (ATCC, USA), 37 ° C. in RPMI 1640 (Gibco, BRL, USA) medium supplemented with 10% fat bovine serum (FBS) and 0.05 mM 2-mercapto ethanol. Cultures in a CO ₂ incubator were used for the experiment.

(2) Free amount measurement of tumor necrosis factor-a (TNF-a)

In order to determine the effect of the composite herbal extract of Example 1 on the tumor necrosis factor-a (TNF-a) glass, tumor necrosis factor-a (TNF) using the cells prepared by the method described in (1) 1 above The free amount of -a) was measured by enzyme immunoassay (ELISA). Specifically, the cells are dispensed at 5 × 10 ⁵ cells / well in a 96-well plate, and the cells are added to liberate tumor necrosis factor-a (TNF-a) by adding lipopolysaccharide (LPS). Activated.

Simultaneous treatment with LPS and the complex herbal extract prepared in Preparation Example 1 in a variety of concentrations incubated for 24 hours.

After 24 hours, the amount of tumor necrosis factor-a (TNF-a) in the cell culture was measured by enzyme-linked immunosorbent assay (ELISA). Absorbance was measured at 450 mm using Dynatech MR-7000 (Dynatech Laboratorie) instrument. Tumor necrosis factor-a (TNF-a) release was calculated as a percentage of the control group (LPS only). It was.

Inhibitory effect of the tumor necrosis factor-alpha (TNF-α) according to the concentration of the mixed herbal medicine extract by the above process is shown in Table 2 below.

As can be seen in Table 2, the cells to which the composite herbal extract of Preparation Example 1 was added, the free TNF-α production rate of the control group at a concentration of 2 mg / ㎖ 8.7%, 4 to 5 times more tumor than the comparative example It was confirmed that the necrosis factor-alpha (TNF-α) glass inhibitory effect was exhibited.

Experimental Example 2 Analysis of Inhibitory Effect of Nitric Oxide (NO) Formation by a Complex Herbal Extract

In order to determine the effect of the composite herbal extract of Preparation Example 1 on the inhibition of nitric oxide (NO) production, which is a mediator of inflammation produced in RAW 264.7 cells, which is a mouse macrophage call line The experiment was performed.

(1) Cell selection and cell culture

In order to evaluate the effect of the composite herbal extract of Preparation Example 1 on the production of nitric oxide, RAW 264.7 cells (ATCC No. TIB-71), a cell line derived from mouse, were purchased from the American Cell Line Bank (ATCC, USA). DMEM (Gibco, BRL, USA) medium to which 10% FBS (fetal bovine serum) was added was cultured in a 37 ℃, CO ₂ incubator used in the experiment.

(2) Nitric oxide (NO) production amount measurement

In order to evaluate the effect of the composite herbal extract of Preparation Example 1 on the nitric oxide production, the amount of nitric oxide production was measured using the cells prepared by the method described in (1). Specifically, cells were activated at 5 × 10 ⁵ cells / well in a 96-well plate. Simultaneously with LPS, the test group was incubated for 12 hours by treating the compound herbal extract of Preparation Example 1 at various concentrations. After 12 hours, the amount of nitric oxide in the cell culture was measured by a Greries reaction.

As in Experimental Example 1, in order to compare the excellent efficacy of the complex herbal extract of Preparation Example 1, the same complex herbal extract of Comparative Examples 1 to 3 used in Experimental Example 1 was used in the experiment.

Inhibitory effect of the nitric oxide production according to the concentration of the composite herbal extract by the above process is shown in Table 3 below.

As can be seen in Table 3, the cells to which the extract of the complex herbal medicine of Preparation Example 1 was superior to the nitric oxide production inhibitory effect compared to the cells of Comparative Examples 1 to 3, in particular at about 6 mg / ㎖ concentration of about 2 to It was confirmed that the effect of inhibiting the production of nitric oxide about 5 times.

Experimental Example 3 Inhibitory Effect of Matrix Metalloproteinase (MMP) Synthesis on Complex Herbal Extracts

To find out the effect of the composite herbal extract of Preparation Example 1 on the synthesis of matrix metallo proteinase-13 (MMP-13) as a matrix depleting substance produced in SW1353 cells that are human chondrosarcoma cell line In order to perform the following experiment.

(1) MMP-13 synthesis amount measurement

In order to evaluate the effect of the composite herbal extract of Preparation Example 1 on the synthesis of MMP-13, SW1353 cells (ATCC No. HTB-94), which is a human-derived cell line, were purchased from the American Cell Line Bank (ATCC. USA). DMEM (Gibco, BRL, USA) medium with% FBS (fetal bovine serum) was incubated in 37 ℃, CO ₂ incubator used in the experiment.

(2) MMP-13 synthesis amount measurement

In order to determine the effect of the composite herbal extract of Preparation Example 1 on MMP-13 synthesis, MMP-13 expression was measured using cells prepared by the method described in (1) above. Specifically, cells were dispensed at 5 × 10 ⁵ cells / well in a 96-well plate, and after 24 hours, PBS buffer (NaCl 8 g, K value 0.2 g, Na ₂ HPO ₄ 1.44 g, KH ₂ PO). ₄ 0.24 g / 1 L distilled water, pH 7.4) and then incubated for 24 hours in DMEM medium without FBS. After treating the complex herbal extract of Preparation Example 1 in the test group for 1 hour, the control group and the test group were treated with tumor necrosis factor-a (TNF-a). After 24 hours, the amount of MMP-13 in the cell culture was measured using Western blotting.

That is, the cell culture was applied to SDS-polyamide gel and electrophoresed, and then transferred to the membrane using a semi-dry blister (Trans-BLOT SD system, Biorad). Membranes were washed once with TBS-T (10 mM Tris-buffered Saline, 0% Tween-20, pH7.2) and left for 1 hour with blocking solution (3% skim milk). After 1 hour, washed twice with TBS-T, and then a single antibody (chemicon) for human MMP-13 was diluted 1: 3000-5000 and reacted for 2 hours. After the reaction, washed three times with TBS-T, HRP-bound goat anti-mouse IgG (Horseradish peroxidase conjugated goat anti-mouse IgG, promega) was diluted and reacted for 1 hour. After 1 hour, the membrane was washed again 3-4 times, and then confirmed using an enhanced chemiluminesence (ECL) western blot analysis system (Amersham).

As in Experimental Example 1, in order to compare the excellent efficacy of the complex herbal extract of Preparation Example 1, the same complex herbal extract of Comparative Examples 1 to 3 used in Experimental Example 1 was used in the experiment. Experimental results of the effect of the composite herbal extract of Preparation Example 1 on the expression of MMP-13 is shown in FIG.

As can be seen in Figure 1, when the composite herbal extract of Comparative Examples 1 to 3 and the composite herbal extract of Preparation Example 1 were treated at the same concentration (6 mg / ㎖), the composite herbal extract of Preparation Example 1 When the composite herbal extracts of Comparative Examples 1 to 3 when compared to the strength of the band was significantly reduced can be confirmed, from the results of the composite herbal extract of Preparation Example 1 was confirmed that the MMP-13 synthesis inhibitory effect is excellent. there was.

Experimental Example 4 Toxicity Test of Complex Herbal Extract of Preparation Example 1

The herbal medicines used in the present invention are classified as food ingredients, so it is determined that there is no problem in the stability of the composite herbal extract of Preparation Example 1, but the present inventors conducted toxicity tests in the following manner to check the stability by oral administration thereof.

Toxicity experiments were carried out using SPF mice, 6 weeks old, as experimental animals. In 10 animals per group, the compound herbal extract of Preparation Example 1 was dissolved in 0.9% Nacl and fastened for 17 hours to mice fed at a dose of 2 g / kg and 5 g / kg. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Blood tests and blood biochemical tests were performed. The necropsy was performed to visually observe the abdominal and thoracic organ abnormalities.

As a result of the test, the herbal extract of Preparation Example 1 had no specific clinical symptoms or dead animals in all the administered animals, and no toxicity change was observed in blood test, blood biochemical test, and autopsy findings.

As a result, the composite herbal extract of Preparation Example 1 of the present invention did not show a toxic change to 5g / kg in the mouse, and the minimum lethal dose (LD ₅₀ ) was confirmed to be a safe substance of 5 g / kg or more.

Experimental Example 5 Effect of Complex Herbal Extracts on the Arthritis Progression Inhibitory Effect in Type 2 Collagen-Induced Arthritis

In order to determine whether the composite herbal extract of Preparation Example 1 of the present invention can inhibit the progression of arthritis, the following experiment was performed.

(1) Preparation of Collagen-Induced Arthritis Mouse Model

In order to make type 2 collagen-induced arthritis experimental animals, male DBA / 1 mice, 5-6 weeks old, were purchased from SCI Co., Ltd. in Japan, and mice were bred under conditions of 21 ° C. and 40% humidity. After dissolving bovine type 2 collagen (Kondrex, Japan) at a concentration of 2 mg / ml in 0.05% acetic acid, bovine type 2 collagen was dissolved in the same amount of complete adjuvant (Kondrex, Japan, Japan). Mixed with. A cool suspension was made using a T-connector connected to a 3 ml syringe while cooling on ice. After confirming that the suspension was well formed, 100 µl of the collagen suspension was injected subcutaneously into the tail of the mouse (courtenay JS, et.al., Nature, 283, pp666-668, 1980).

(2) Oral administration of the complex herbal extract

The composite herbal extract prepared in Preparation Example 1 was dissolved in water and prepared by filtration using a 0.25 uM filter. The prepared herbal extracts were administered once daily at 276 mg / kg / day and connected directly to the mouth of the mouse by connecting the maximus to a 1 ml syringe for 3 weeks.

In order to compare the excellent efficacy of the complex herbal extract of Preparation Example 1, penicillamine (penicillamine), which is a therapeutic agent for arthritis, was administered as a positive control.

(3) arthritis Progress  observe

The invention of arthritis in mice injected with collagen suspensions is known to occur 30 days from the day of injection. Visual observation of arthritis lesions was performed using the following scores based on the above references.

0: No swelling and seizures, 1: slight redness and swelling of the joint, 2: clear redness and swelling of the joint, 3: estrus, including the limb joint, severe swelling, 4: swelling in all areas of the joint This is extreme.

Therefore, the highest score of joint lesion is 16, including the left and right front and back per mouse, and the highest score per leg is 4 (Courtenay J. S., Dallman M. J., Dayan AD, et al .: Immunisation against heterologous

type II collagen induces arthritis in mice. Nature 283: 666-668. 1980).

(4) Observation of Arthritis Progression Inhibitory Effect in Type 2 Collagen-Induced Arthritis Experiment Animals

The therapeutic effect of inhibiting the progression of arthritis after oral administration of the composite herbal extract prepared in (2) to a type 2 collagen-induced arthritis experimental animal is shown in Table 4 below.

As a result, as can be seen in Table 4, when the composite herbal extract of Preparation Example 1 of the present invention was administered, it was confirmed that the efficacy of inhibiting the pathological progression of arthritis than the known arthritis treatment agent so far.

Preparation Example 1 Preparation of Tablet

200 mg complex herbal extract of Preparation Example 1

Lactose 100 mg

Starch 100 mg

Magnesium stearate appropriate amount

According to the conventional tablet manufacturing method, the above-mentioned mixed ingredients are mixed, tableted and tablets are prepared.

Preparation Example 2 Preparation of Capsule

100 mg of the composite herbal extract of Preparation Example 1

Lactose 50 mg

Starch 50 mg

Talc 2 mg

Magnesium stearate appropriate amount

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Preparation Example 3 Preparation of Liquid

1000 mg complex herbal extract of Preparation Example 1

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Purified water was added to adjust the total volume to 1000 ml. According to a conventional method for preparing a liquid, the above components are mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

[ Formulation example  4] Health drink  Produce

1000 mg complex herbal extract of Preparation Example 1

2 g liquid fructose

100 g oligosaccharides

2 g of sugar

1 g of salt

900 ml of water

After mixing the above components according to the conventional healthy beverage production method, and then stirred and heated at 87 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 ml container, sealed sterilized and stored in the refrigerator and then Used to prepare the healthy beverage composition of the invention.

Claims (8)

A pharmaceutical composition for the treatment and prevention of arthritis, which contains a complex herbal extract including goldamcho, jinkyo, Haedongpi, tofu, windbreak, gastrointestinal tract, hyssop, and Seokchangpo as an active ingredient. The method of claim 1,
The complex herbal extract is a compounding weight ratio of goldamcho, jinkyo, Haedongpi, Tokchung, windbreak, gastric, hyssop, and Seokchangpo of 1: 0.5 to 3: 0.05 to 1: 0.5 to 3: 0.5 to 2.5: 0.5 to 3: 0.5 to 3 : Pharmaceutical composition, characterized in that 0.5 to 3. The method of claim 2,
The composite herbal extract is dried after the combined herbal medicine of goldamcho, jinkyo, Haedongpi, Toechung, Windbreak, Gasyeongseon, Woo Seol, and Seokchangpo, water of 5 to 20 times the volume of the combined herbs, lower alcohol having 1 to 4 carbon atoms or their Adding a mixed solvent and repeatedly extracting 2 to 5 times by hot water extraction for 1 to 24 hours at an extraction temperature of 80 to 100 ° C. to obtain an extract soluble in a solvent; And
After filtering the extract obtained, and then concentrated under reduced pressure at 40 to 80 ℃ to prepare a powder extract through lyophilization or vacuum drying; pharmaceutical composition comprising a. The method of claim 1,
The active ingredient is a pharmaceutical composition, characterized in that 0.001 to 95% by weight of the total composition. The method of claim 1,
The arthritis includes acute and chronic rheumatism; Atrophic arthritis; Chronic inflammatory arthritis; Collagen-induced arthritis; Deformable arthritis; Infectious arthritis; Menopausal arthritis; Break arthritis; Hypertrophic arthritis; Purulent arthritis; And arthritis and degenerative osteoarthritis diseases caused by tuberculous arthritis. 6. The method of claim 5,
The arthritis is a pharmaceutical composition, characterized in that at least one selected from acute rheumatism, chronic rheumatism, collagen-induced arthritis and chronic inflammatory arthritis. 7. The compound according to any one of claims 1 to 6,
The pharmaceutical composition is a pharmaceutical composition, characterized in that to prevent or treat arthritis through the inhibition of the release of tumor necrosis factor, the synthesis of matrix metalloproteinase-13, and the inhibition of the production of nitric oxide. Health functional food for preventing and improving arthritis, which contains a complex herbal extract including goldamcho, jinkyo, Haedongpi, tofu, windproof, gastrointestinal, hyssop, and Seokchangpo.

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