KR101454336B1 - Compositions for preventing and treating arthritis - Google Patents

Abstract

The present invention relates to a composition for improving or treating an inflammatory disease comprising an extract of natural complex obtained by mixing dried and appropriate ratio of Juniper berry, European grape skin, European grape seed, White rice, And a method for producing the same. Further, the present invention provides a health functional food composition for preventing or ameliorating osteoarthritis containing any one of the above extracts as an active ingredient.
The extract of the present invention has an inhibitory activity on the production of nitrogen oxides (NO), inhibition of inflammatory cytokines tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and prostaglandin E ₂ (PGE ₂ ) And exhibits an effect for the improvement and treatment of inflammatory diseases. Since it has no cytotoxicity, it can be effectively used for medicines and health functional foods for the improvement and treatment or prevention of inflammatory diseases.

Description

Compositions for preventing and treating arthritis [

The present invention relates to a functional food and a natural substance new drug composition having an effect on prevention and treatment of arthritis. More specifically, the present invention relates to a composition for prevention and treatment of osteoarthritis comprising juniper berry, European grape skin, European grape seed, And to a health functional food composition.

In recent years, life expectancy is likely to increase to 85 years or older within the next 20 years due to the improvement in people's standard of living and the development of life sciences and medical technology. In 2010 and beyond, 10 to 15% And it is expected to become an aging society in earnest. As the elderly population increases, the number of patients with degenerative diseases such as dementia, arteriosclerosis and arthritis is increasing. This causes economic and psychological difficulties for themselves and their families, and it is becoming a social problem.

Although the market for drugs and functional foods related to degenerative diseases is rapidly increasing due to the increase in the elderly population, there is still a lack of development of essential therapeutic or preventive medicines for degenerative diseases. Prevention and treatment of aging and related diseases are mainly devoted to drugs and surgery, and there are few health functional foods in terms of prevention. Therefore, it is necessary to develop a preventive food for aging disease which has functional and effect using food-grade natural substance which is secured from the clinical aspect, and the interest of such aging-prevention food is increasing with the increase of the elderly group .

Development of functional food materials and products has been promoted in medicinal and edible resources that have been used in the private sector due to the development of life sciences and has been rapidly spreading to industry. Especially recently, as Korea has entered into an aging society, 80% Most of these cases are known to have osteoarthritis. Arthritis, a typical degenerative disease, is known to have at least 600,000 patients in Korea, and the world has the largest number of treatments for osteoarthritis (osteoarthritis, osteoporosis).

Degenerative diseases such as osteoarthritis are increasing with age and eating habits. Despite the development of various treatments, the aging population and western eating habits still remain serious diseases. The mortality rate is low, Which is a major factor in lowering the quality of life. The evaluation and study of the efficacy of physiologically active substances for the improvement of degenerative diseases and the development of functional foods are expected to accelerate the commercialization and commercialization of more functional food candidates. These diseases are caused by environmental factors such as genetic factors and dietary habits , It is very meaningful to study functional materials having physiological activity contained in biomaterials because they can increase immunity by steadily ingesting food having a preventive effect.

According to the 2008 Korea Food & Drug Administration (KFDA), health food products sold in Korea in 2007 accounted for a total of KRW 723.4 billion, of which red ginseng products accounted for 45.2%, totaling KRW 327.0 billion, followed by aloe products (KRW 79.7 billion), vitamin / 78.5 billion won), ginseng products (35 billion won), and glucosamine-containing products (26.9 billion won). In particular, glucosamine-containing products are steadily being sold to the elderly. However, there is a disagreement between the medical community and the industry about whether glucosamine is really effective in preventing arthritis. Manufacturers argue that glucosamine promotes cartilage protein production by promoting the production of cartilage proteins, but most physicians claim that they have analgesic effects that alleviate joint pain, but no evidence of arthritis prevention or treatment. .

Recently, studies for discovering arthritis-related functional foods and natural product drugs from natural products that have been used as traditional medicines or folk remedies have received worldwide attention, particularly in developed countries such as the United States, Europe, and Japan. Interest in the world's leading pharmaceutical companies has been rising to promote the development of health functional foods and natural products from relatively stable and highly effective natural foods.

All inflammatory diseases, including osteoarthritis, are manifestations of various types of infection and pre-in vivo defense against irritants in vivo metabolites, and various chemical mediators are involved in the mechanism of inflammatory expression , The pathogen is also very complicated. Conventional anti-inflammatory agents are classified into steroidal and non-steroidal anti-inflammatory agents. Most of synthetic anti-inflammatory agents are accompanied with various side effects in addition to the main action. Therefore, there is a desperate need for the development of anti- It is true.

Osteoarthritis is a degenerative disease that occurs in older age groups with decreased metabolism and cell regeneration ability. Its basic treatment is almost impossible, and surgery, medication and physical therapy are performed. However, except for some adjuvant therapies, You have to take risks.

Osteoarthritis is characterized by gradual degenerative changes in the joints, often involving weight bearing joints. Osteoarthritis is also the most common form of synovial arthritis occurring in synovial joints (McAlindon TE et al., Am J Med 106, pp 151-157, 1999). Knee arthritis is the most common symptom of arthritis associated with osteoarthritis and is a weight-bearing joint, which affects exercise and directly affects quality of life (Felson DT et al., Arthritis Rheum. -918, 1987).

Symptoms are more common in women, with an increase in the number of symptoms and an increased incidence of symptoms in 7% of the population aged 65 to 70 years and in 11.2% of those aged 80 years or older. Osteoarthritis is diagnosed in 27.4% of the population aged 70 to 70 years and 43.7% of the population aged 80 years or older (Dougados M et al., J Rhemmatol. 19 (3), pp 378-84, 1992). Approximately 10% of people aged 60 or older feel uncomfortable with degenerative arthritis. In the United States, 20 million people are affected by quality of life, and more than $ 60 billion annually is used to treat arthritis (Buckwalter JA et al., Clin Orthop Relat Res. 427S, pp6-15, 2004).

Risk factors for degenerative knee osteoarthritis with radiographic degeneration other than increased age include family history, genetic problems that can affect skeletal or joint growth and morphogenesis, joint damage, specific repetitive movements, obesity (Dieppe PA et al., Lancet, 365, pp 965-973, 2005; Felson DT., Clin. Orthop Relat Res. 427S, pp 16-2, 2004). It has been reported that synovitis around the joints plays an important role in joint pain, joint inflammation and cartilage damage. Prostaglandin E2, PGE2, nitric oxide (NO) (Brenner SS et al., Osteoarthritis Cartilage. 12, pp 469-475, 2003).

Osteoarthritis in the modern age is a common and generalized disease due to the rapid increase of obese patients due to changes in eating habits and traffic, but there is no selective treatment drug because the mechanism of arthritis is not known yet. Therefore, the purpose of treatment of arthritis is to reduce pain and inflammation of the joints, and to prevent deformation of the joints. Because there is no fundamental treatment for osteoarthritis, physical and exercise therapy is performed as the basic treatment method, and appropriate nonsteroidal antiinflammatory drugs (NSAIDs) are used to alleviate the inflammation. If inflammation is severe, gold salt therapy or penicillamine ). If the above method is not effective, a steroid preparation is used. In the case of refractory, an immunosuppressive agent is used. Nonsteroidal anti-inflammatory drugs (NSAIDs) used for anti-inflammatory and analgesic effects on arthritis mainly inhibit cyclooxygenase and inhibit the production of prostaglandins (inflammation) . Among them indomethacin and phenylbutazone have strong anti-inflammatory effects compared to other NSAIDs. Indomethacin is one of the most potent inhibitors of inflammation-inducing prostaglandin production, and is well absorbed by oral administration, but these drugs have been associated with dizziness, delirium, and, in rare cases, with hallucinations, as well as side effects on the common gastrointestinal tract Psychosis has been reported to be so toxic that 33% of patients at high dose have many side effects such as discontinuation of medication. Phenylbutazone has also been used effectively in the treatment of rheumatoid arthritis, ankylosing spondylitis and acute gouty arthritis compared to other early NSAIDs, but is currently not used due to severe toxicity including hematologic abnormalities such as granulocytopenia or aplastic anemia. Compared to these NSAIDs, steroids are the fastest method available to patients, and they are known to be a drug that shows rapid and dramatic anti-inflammatory effects and pleasure to the patient. However, as is widely known, this drug is less resistant to bacterial infections and causes worsening diabetes, Cushing's syndrome, adrenal insufficiency, mental dysfunction, and is very toxic, It is known that it is difficult to use, and it should be avoided if possible. Conventional treatments for joint disease have serious side effects and are less effective, consumers are increasing their preference for functional foods with fewer side effects. Glucosamine-containing products, which are regularly sold to the elderly, have few side effects, but the medical community alleviates joint pain However, since there is no evidence that arthritis prevention or therapeutic effects have been proven, the search for new functional materials that are backed by efficacy will become an aging society, and there will be a surge in demand for bone disease-improving products as well as interest in joint diseases It is expected.

The present inventors have searched for useful ingredients from traditional biological materials based on folk remedies that have been used for a long time without adverse effects and have prepared traditional herbal composition processed foods As a result of intensive researches to improve the competitiveness of functional material technologies and to develop individual medicinal certification methods for useful ingredients of traditional materials, we have found that the use of Juniper berry, European grape skin, European grape seed And a pharmaceutical composition and a food composition for the prevention and treatment of inflammation-related diseases which contain white rice, wheat, and ginseng as active ingredients.

Juniper berry is said to have dried ripe berries from Juniperus rigida Sieb. Dt Zucc.), An evergreen tree belonging to the family Porphyra, according to prehistoric records. Let urine come out well. In pharmacological experiments, it has been shown that it works in the form of dry matter, gadolinium, and bacillus, and is used for edema, bladder and urethral diseases, rheumatoid arthritis and amoebic dysentery.

All grapes, including European grapes, contain antioxidants. Grape seed extracts contain a large amount of proanthocyanidins, a type of bioflavonoid, which improve the function of the connective tissues and improve cardiovascular disease, arthritis and cancer. It prevents tissue damage by eliminating the generator oxygen that is triggered. In addition, proanthocyanidin in grape seeds and skin has antiviral, antibacterial, anti-inflammatory and anti-allergic properties. The grape seed extract can be effective for about 3 days in the human body, and the effect is 20 times of vitamin C and 50 times of vitamin E.

White rice is the dried root of Cynanchum atratum Bunge and C. versicolor Bunge (Aspergillus and Asclepiadaceae). Appearance is light yellow-brown, elongated roots gathered in short rootstock, length 10 ~ 25㎝, diameter 1 ~ 2㎜. The roots are easy to break, have a peculiar smell, and taste a bit spicy. Steroid glycosides, Neolignan, and Aetophenone have been identified. Their pharmacological actions include anti-cancer, anti-inflammatory and antipyretic effects, diuretics, fever and fever of fever , And limb swelling in stroke patients.

It is dried with the roots of Achyranthes japonica. Two sprouts are used. The root of the sugarcane contains triterpenoid saponin and contains a large amount of potassium salt. It has been known for a long time that blood circulation, diuretic effect, blood pressure lowering effect and analgesic action are known, and it is known that it has an effect of interpolating, watching and strengthening muscular strength (Jung Hyun Suh, Yeonglim Corp, pp340-341, 1998) .

It is a perennial plant that is grown in Central China and origin in China. In the present invention, the rhizome is dried and used. Typical examples thereof are Anemarrhena asphodelorides BUNGE, which is used for anti-inflammation, fever, diarrhea, diuretic, back pain and sedation in the pharmacopoeia (KP, JP). This medicinal plant contains steroid sapogenins, flavonoids and tannins, such as sarsasaponin and 2-hydroxy sarsasapogenin, as well as 6% asphonin. In the case of Dong-Bo-bo-gyung and Hwa-gok gangmyeon, the hair was used for osteoporotic fever. The interpretation of the term osteoporosis means that the osteoporosis is manifested in the bone and the patient complains of chronic pain.

Accordingly, the inventors of the present invention prepared a natural complex extract of Juniper berry, European grape skin, European grape seed, White rice, Wasser and Zymo, and found that the extracts of the present invention inhibit the production of nitrogen oxides (NO), tumor necrosis factor (TNF-α), interleukin-1 beta (IL-1β) and prostaglandin E ₂ (PGE ₂ ), and thus has excellent prophylactic or therapeutic effects against inflammatory diseases and has no cytotoxicity To complete the present invention.

According to a preferred embodiment of the present invention for solving the above problems, there is provided a herbal composition which is effective for preventing osteoarthritis comprising, as an active ingredient, a natural compound extract comprising Juniper berry, European grape skin, European grape seed, White rice, .

Further, the present invention provides a health functional food composition for preventing or ameliorating osteoarthritis containing any one of the above extracts as an active ingredient.

The present invention relates to a pharmaceutical composition containing, as an active ingredient, any one selected from the group consisting of Juniper berries, European grape skin, European grape seed, natural extract consisting of white rice, Or a pharmaceutically acceptable salt thereof. The present invention also provides a composition for preventing or treating an inflammatory disease.

According to a preferred embodiment of the present invention, the amount of the extract is 0.01 to 95% by weight of the total composition.

According to another preferred embodiment of the present invention, the natural complex extract is mixed with a composition ratio of 1: 1: 1: 1: 1: 1: 1 ratio of juniper berry, European grape skin, European grape seed, .

The present invention also provides a health functional food for the prevention or treatment of inflammatory diseases containing the above composition as an active ingredient.

According to a preferred embodiment of the present invention, the health functional food comprises a foodstuff acceptable food additive and is in the form of any one of tablet, capsule, pill or liquid, and is in the form of food, beverage, gum, tea, Vitamin complex, and health functional food.

The present invention also relates to a method for producing a nutritional composition, comprising the steps of: drying each natural product of Juniper berry, European grape skin, European grape seed, White rice, Adding the dried natural products at the same weight ratio, adding 5 to 20 times volume or weight of the natural product, extracting the product for 2 to 5 times at a temperature of 80 to 100 캜 for 3 to 5 hours by hot water extraction; Filtering the extract obtained from the step, concentrating the extract at a reduced pressure at 40 to 80 ° C, and azeotropically concentrating the obtained extract 1 to 5 times with 10 to 60 times the total amount of the extract; And preparing an extract of the azeotropic mixture as a powdery extract through lyophilization or vacuum drying. The present invention also provides a method for preparing a composition for preventing and improving osteoarthritis.

The present invention relates to a composition for improving or treating an inflammatory disease, which comprises a natural compound extract consisting of Juniper berry, European grape skin, European grape seed, white rice, (TNF-α), interleukin-1 beta (IL-1β), and prostaglandin E ₂ (PGE ₂ ) Since it has no cytotoxicity, it can be usefully used for medicines and health functional foods for the improvement and treatment or prevention of inflammatory diseases.

FIG. 1 is a data obtained by measuring the cytotoxicity of RAW 264.7 macrophage cell line derived from BALB / c mouse using the natural compound extract of the present invention.
FIG. 2 is a data obtained by measuring the inhibitory effect of the natural combination extract of the present invention on the production of nitrogen monoxide (NO) by RAW 264.7 macrophage cell line derived from BALB / c mouse.
FIG. 3 is a graph showing the inhibitory effect of the natural compound extract of the present invention on tumor necrosis factor (TNF-α) production in RAW 264.7 macrophage cells derived from BALB / c mice.
FIG. 4 is a data obtained by measuring the inhibitory effect of the natural compound extract of the present invention on the production of interleukin 1-beta (IL-1β) by RAW 264.7 macrophage cell line derived from BALB / c mouse.
FIG. 5 shows experimental results of inhibiting the production of prostaglandin E ₂ (PGE ₂ ) by the natural combination extract of the present invention in RAW 264.7 macrophage line derived from BALB / c mouse.

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition for the prevention or treatment of inflammatory diseases containing, as an active ingredient, any one or more of the above extracts selected from the group consisting of Juniper berries, European grape skin, European grape seed, Lt; / RTI >

The extract can be used without limitation such as collected, cultured, or marketed. For example, it is preferable to collect the extract and wash it with water to remove foreign matter and dry it.

The present invention also relates to a method of treating inflammation, comprising administering an extract of at least one selected from the group consisting of Juniper berry, European grape skin, European grape seed, rice white, Prevention or amelioration of a disease or condition.

In this case, the amount to inhibit inflammation is not particularly limited, but is preferably 0.1 to 500 mg / kg, more preferably 1 to 100 mg / kg. The dose may vary depending on the weight, age, sex, health condition, diet, administration period, administration method, elimination rate, severity of disease, etc. of the specific patient.

In order to determine the effect of at least one extract selected from the group consisting of Juniper berry, European grape skin, European grape seed, Japanese white rice, oyster and Japanese oyster according to the present invention for the prevention or amelioration of inflammatory diseases, RAW 264.7 Inhibition of nitric oxide (NO) production in macrophage cell lines, inhibition of inflammatory cytokines TNF-α, interleukin-1 beta, prostaglandin E2 (PGE2) Experiments were performed.

The results of the anti-inflammatory physiological activity tests on the natural compound extracts of Juniper berry, European grape skin, European grape seed, White rice, Wasser and Zymo of the present invention are shown in FIGS.

Accordingly, the present invention provides a natural combination extract consisting of Juniper berry, European grape skin, European grape seed, White rice, Wasser and Zymo, which is effective for inhibiting NO production, inhibiting inflammatory cytokine tumor necrosis factor- Inflammatory diseases, such as edema, dermatitis, allergies, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sphincteritis, and the like, which exhibit an inhibitory effect on the production of IL-1 beta (IL-1?) And prostaglandin E2 Osteoarthritis, rheumatoid arthritis, shoulder inflammation, tendonitis, hay fever, tendinitis, myalgia, osteoarthritis, arthritis, osteoarthritis, osteoarthritis, osteoarthritis, osteoarthritis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, , Drugs for preventing or improving hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis and acute or chronic inflammatory diseases, Can be usefully used.

Also provided is a cartilage regeneration effect comprising the extract of the present invention and a pharmaceutically acceptable food additive, and a health functional food for improving inflammation and pain. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Examples of the foods to which the compound of the present invention can be added include various foods, beverages, gums, tea, And health functional foods. In detail, the present invention provides a cartilage regeneration effect and an anti-inflammatory and analgesic health functional food comprising an extract containing the extract of the present invention as an active ingredient and a pharmaceutically acceptable food-aid additive.

The pharmaceutical composition of the present invention may be various oral or parenteral formulations. 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 one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.

Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

According to a preferred embodiment of the present invention, it is preferred that the amount of extract has a weight ratio of 0.01 to 95% of the total composition.

According to a preferred embodiment of the present invention, it is preferable that the composition ratio of juniper berry, European grape skin, European grape seed, White rice, Wasser and Zymo are mixed in a weight ratio of 1: 1: 1: 1: 1: 1.

According to a preferred embodiment of the present invention, each natural product of Juniper berries, European species grape skin, European species grape seed, white rice, wool and plum is dried, and then 5 to 20 times by volume / The extract is extracted with water for 2 to 5 times by hot water extraction at an extraction temperature of 80 to 100 ° C for 3 to 5 hours. The extract obtained from the above step is filtered and concentrated under reduced pressure at 40 to 80 ° C. And a second step of azeotropically concentrating the extract concentrate 1 to 5 times with 10 to 60 times the total amount of extract and then lyophilizing or vacuum drying the extract to prepare a powdery extract, and a pharmaceutical composition for preventing and improving osteoarthritis Of the present invention.

Hereinafter, the present invention will be described in detail.

However, the following examples, reference examples and experimental examples are illustrative of the present invention, and the present invention is not limited thereto.

Example 1. Preparation of natural herbal medicine extracts for prevention and improvement of osteoarthritis according to the present invention

1-1. Composition of natural complex herbal medicine

In this Example, a natural combination herbal composition for prevention and improvement of osteoarthritis was prepared by the composition of the natural combination herbal medicine shown in Table 1 below.

designation Scientific name ratio (%) Juniper Berry Juniperus communis 16 European species grape skin Vitis vinifera 16 European grape seed Vitis vinifera 16 White rice Cynanchum atratum 16 The Achyranthes japonica 18 Ji anemarrhena asphodeloides 18

1-2. Natural herbal medicine extract (hot water extract)

According to the ratio shown in Table 1, 16% by weight of Juniper berries, 16% by weight of European seed grains, 16% by weight of European grape skin, 16% by weight of white rice, 18% by weight of astaxanthin and 18% . Dried water is added to natural products at a ratio of 5 to 20 times volume / weight of natural product, and the product is dried at an extraction temperature of 80 to 100 ° C for 3 to 5 times For 2 to 5 times. The extract obtained from the above step was filtered, and then concentrated under reduced pressure at 40 to 80 ° C.

Reference Example 1. Preparation of experimental material

(Dulbecco's Modified Eagle Medium, Cat # 11995, hereinafter referred to as 'DMEM') and fetal bovine serum (Cat # 16000-044, hereinafter referred to as 'FBS') which is a cell culture medium. , And Streptomycin-penicillin (Cat # 15140-122) were purchased from Gibco BRL (NY, USA). The Aqueous One Solution Cell Proliferation Assay (MTS) kit and the Griess Reagent System (Cat # G3580) were purchased from Promega (WI, USA). An ELISA kit (TNF-α, Cat # 88-7324; IL-1β, Cat # 88-7013) for cytokine measurement in cell culture solutions was purchased from eBioscience Inc. (CA, USA) And the PGE ₂ assay kit (Cat # SKGE004B) was purchased from R & D Systems, Inc. (MN, USA). All reagents used in the experiment were used for analytical grade or higher.

Reference Example 2. Cell culture

RAW264.7 cells (Korean Cell Line Bank (KCLB)), a macrophage cell line of mouse, was cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin under the conditions of 37 ° C and 5% CO ₂ And used in the following experiments. In addition, all the test results were expressed as means and standard deviation. The significance test was performed by Student's t-test (p <0.001) using Student's t-test (Sigma Plot, version 7.0 for Window) Or Lipopolysaccharide (hereinafter referred to as 'LPS'), which is an inflammation-inducing component.

Experimental Example 1. MTS analysis

To determine the toxicity of the extracts obtained in Example 1-2 above to cells, Desai, A, Vyas T, Amiji M. Cytotoxicity and apoptosis enhancement in brain tumor cells coadministration of paclitaxel and ceramide in nanoemulsion formulations J (3-carboxyphenyl) -2H-tetra-zolium inner (3-carboxyphenyl) -2H-tetrazolium salt described in salt, hereinafter referred to as &quot; MTS &quot;).

RAW 264.7 cells cultured by the method of Reference Example 2 were dispensed into a 96-well plate at 1 × 10 ⁴ cells / well, and the extracts obtained in Example 1 were diluted with 5, 10, 20 , 50 [mu] g / ml) for 24 hours. After adding 20 μl of MTS solution per well, the reaction was carried out in a 5% CO ₂ incubator at 37 ° C. for 4 hours. The absorbance at 450 nm was measured using a microplate reader (ELISA reader, Tecan, Austria) Cell viability for the untreated control was expressed as a percentage. In order to calibrate the absorbance of the medicinal materials at each concentration, the medium in which the cells were removed was cultured together, and the absorbance of the control group and the experimental group was compared and expressed, and the cell survival rate was expressed as a percentage (mean value ± standard deviation).

As shown in FIG. 1, the natural herbal medicine extract of Example 1-2 was treated with each concentration (5, 10, 20, 50 ug / ml) for 24 hours. As a result, / Ml, cell survival rate of 90% or more was observed. Celecoxib group showed cell survival rate of 74 ~ 80%.

Experimental Example 2. Measurement of nitrogen monoxide (NO) production amount

To measure the amount of nitric oxide (NO) produced, a method described in Wang S et al., J. Ethnopharmacol., 114 (3), pp 458-462, 2007, Respectively. RAW264.7 cells cultured by the method of Reference Example 2 were pre-treated with the herbal extracts obtained in Example 1-2 and treated with 1 ng / ml of LPS for 1 hour and cultured for 24 hours. The same amount of grease reagent as that of the culture solution (50 μl) was added, and the reaction was carried out at room temperature for 10 minutes. Then, the absorbance was measured at 540 nm using an ELISA reader. The concentration of nitrogen monoxide in the culture medium was determined using a standard curve of the concentration of sodium nitrite.

As shown in FIG. 2, when the concentration of LPS was 1 ng / ml, the amount of nitrogen monoxide (NO) was increased about 36 times as compared with the control without any treatment. When the concentration of 50 ㎍ / ㎖ of complex herbal extracts was pretreated for 1 hour, the reduction rate of the production of LPS - induced NO was about 62% at the low concentration of 5 ug / ml and the concentration of the high concentration of 50 ㎍ / And 75%, respectively.

Experimental Example 3. Measurement of Mouse TNF-α, mouse IL-1β cytokine production

As an indicator of inflammation, an immunoenzymatic assay kit was used to measure the amount of proinflammatory cytokine tumor necrosis factor-alpha (mouse TNF-α) and interleukin-1 beta (mouse IL-1β) And then subjected to ELISA (Enzyme-Linked Immunosorbent Assay).

The RAW264.7 cells cultured by the method of Reference Example 2 were pretreated with the natural herbicidal extracts obtained in Example 1 at concentrations of 5, 10, 20 and 50 ug / ml for 1 hour, and then treated with 1 ng / ml LPS was treated. After culturing for 24 hours at 5% CO ₂ at 37 ° C, the cell culture was diluted to an appropriate concentration. 100 [mu] L was added to a 96 well plate coated with each cytokine capture antibody, and the plate was allowed to stand overnight at 4 [deg.] C. 100 μl of each cytokine biotinylated antibody reagent was treated in each well and reacted at room temperature for 1 hour, followed by 5 times washing with washing buffer After washing, streptavidin-HRP solution (100 μl) was added to each well, reacted at room temperature for 1 hour, and then washed 5 times with washing buffer. 100 μl of di (2-ethylhexyl) -2,4,5-trimethoxybenzalmalonate (TMB) substrate was treated with 5 ~ 30 After incubation for one minute, 100 μl of stop solution was treated and absorbance was measured at 450 nm.

As shown in FIG. 3, the amount of tumor necrosis factor-alpha (TNF-α) induced by LPS in the treatment group of low concentration of 5 μg / ml and a high concentration of 50 μg / 39% and 63%, respectively. In addition, as shown in FIG. 4, the amount of IL-1β induced by LPS was 40% at the low concentration of 5 ㎍ / ml and at the high concentration of 50 ㎍ / , And 67%, respectively.

The preparation examples of the composition containing the herbal extract of the present invention will be described below, but the present invention is not intended to be limited thereto but is specifically explained.

Experimental Example 4. Measurement of PGE ₂ production amount

In order to measure the amount of PGE ₂ , another indicator of inflammation, an experiment was conducted using the commercial competitive enzyme immunoassay kit (R & D systems, MN) as follows.

The RAW264.7 cells cultured by the method of Reference Example 2 were pretreated with the natural herb extracts obtained in Example 1-2 at concentrations of 5, 10, 20 and 50 ug / ml for 1 hour, Ml &lt; / RTI &gt; of LPS. After 24 hours, the cell culture solution is injected into a 96-well plate coated with goat anti-mouse in an amount of 100 μl of each culture. 50 μl of the primary antibody solution and 50 μl of PGE ₂ conjugate were added thereto, and the mixture was allowed to stand overnight at 4 ° C. After washing 5 times with washing buffer, 200 μl of substrate solution was treated for 5 ~ 20 minutes, 50 μl of stop solution was treated, and absorbance at 450 nm Were measured.

As shown in FIG. 5, the amount of PGE ₂ produced was about 17 times higher than that of the control without any treatment at a concentration of 1 ng / ml of LPS. In addition, the experimental group treated with the natural herb extract of Example 1-2 showed a reduction effect of 53% at the low concentration of 5 ㎍ / ㎖ and a reduction effect of 88% at the high concentration of 50 ㎍ / ㎖.

Formulation Example 1. Preparation of powder

Herbal medicine extract 20mg

Lactose 100mg

Talc 10mg

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

Formulation Example 2. Preparation of tablets

Complex herbal medicine extract 10mg

Corn starch 100 mg

Lactose 100mg

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

Complex herbal medicine extract 10mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

0.2 mg of magnesium stearate

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation Example 4. Preparation of injection

Complex herbal medicine extract 10mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO _{4 12} H ₂ O 26 mg

It is usually prepared by the above-mentioned component content per 1 ampoule (2 ml) according to the method of injection preparation.

Formulation Example 5. Preparation of a liquid preparation

Herbal medicine extract 20mg

10g per isomerization

Mannitol 5 g

Purified water titration

Each component is added to purified water in accordance with the usual preparation method of the liquid preparation and dissolved, and the lemon flavor is added in an appropriate amount. Then, the above components are mixed, and purified water is added to adjust the total volume to 100 ml. The solution is filled in a brown bottle and sterilized to prepare a liquid preparation .

Formulation Example 6. Preparation of the Ring

Complex herbal medicine extract 1000mg

Lactose 1500mg

Glycerin 1000 mg

Xylitol 500mg

After the above components are mixed, they are prepared to be 4000 mg per one ring according to a conventional method.

Preparation Example 7. Preparation of granules

Complex herbal medicine extract 150mg

Soybean extract 50mg

200 mg of glucose

Starch 600mg

After mixing the above components, 100 ml of 30% ethanol is added and the mixture is dried at 60 ° C to form granules, which are then filled into a capsule.

Formulation Example 8. Preparation of tea bag

The compositions of the herbal extracts were washed with clean water and then steamed for 1 hour at 120 ° C. in a high pressure steam sterilization pot. The moisture content of the above-mentioned six herbals was measured by the atmospheric pressure heating drying method, and the moisture content of the above-mentioned six herbal medicines was measured by drying at a temperature of 80 [deg.] C for about 3 hours in the dried juniper berry, European grape skin, European grape seed, , It is confirmed that all the moisture content is maintained at 5% or less.

160 g of Juniper berries, 160 g of European species grape skin, 160 g of European grape seeds, 160 g of white rice, 180 g of oyster and 180 g of gum are mixed together and pulverized to a particle size of 1 to 2 mm through a pulverizer. After mixing well for 10 minutes or longer, 20 g of the pulverized mixed raw material is put in a hopper for packing the tea bag and packed to complete the tea bag.

Formulation Example 9. Preparation of Health Functional Foods

Complex herbal medicine extract 1000mg

Vitamin mixture quantity

70 비 of vitamin A acetate

Vitamin E 1.0mg

0.13 mg of vitamin B1

0.15 mg of vitamin B2

Vitamin B6 0.5mg

0.2 g of vitamin B12

Vitamin C 10mg

10 μg of biotin

Nicotinic acid amide 1.7 mg

50 엽 of folic acid

Calcium pantothenate 0.5mg

Mineral mixture quantity

1.75 mg ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Calcium phosphate diphosphate 55 mg

Potassium citrate 90mg

Carbonate knife? 100 mg

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a component suitable for a health functional food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above components may be mixed , Granules may be prepared and used in the manufacture of a health functional food composition according to a conventional method.

Formulation Example 10. Preparation of Health Drink

Complex herbal medicine extract 1000mg

Citric acid 1000mg

Oligosaccharide 100 g

Plum concentrate 2g

Taurine 1g

Total purified water 900ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The resulting solution was filtered and sterilized in a sterilized 2 L container, &Lt; / RTI &gt;

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 (6)

Wherein the active ingredient is a natural combination extract comprising Juniper berries, European species grape skin, European species grape seeds, white rice, whiskey, and radish.
The method according to claim 1,
Wherein the amount of the natural complex extract has a weight ratio of 0.01 to 95% of the total composition.
The method according to claim 1,
Wherein the natural combination extract is mixed at a weight ratio of 1: 1: 1: 1: 1: 1 in the composition ratio of juniper berry, European grape skin, European grape seed, white rice,
An anti-inflammatory health functional food comprising the composition of any one of claims 1 to 3 as an active ingredient.
5. The method of claim 4,
The health functional food comprises a food acceptable food additive and is in the form of any one of tablets, capsules, pills or liquids, and is in the form of food, beverage, gum, tea, vitamin complex, &Lt; / RTI &gt; further comprising an anti-inflammatory health functional food.
Drying each natural product of Juniper Berry, European grape skin, European species grape seed, white rice, whisker, and brine;
Adding the dried natural products at the same weight ratio, adding 5 to 20 times volume or weight of the natural product, extracting the product for 2 to 5 times at a temperature of 80 to 100 캜 for 3 to 5 hours by hot water extraction;
Concentrating the extract obtained by concentrating under reduced pressure at 40 to 80 ° C by azeotropic distillation 1 to 5 times with 10 to 60 times the total volume of the extract; And
The method for preparing a composition for prevention and remedy of osteoarthritis according to claim 1, wherein the extract is prepared by freeze drying or vacuum drying.

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