KR101496277B1 - Composition for treating and preventing rheumatoid arthritis - Google Patents

Abstract

In the present invention, disclosed is a composition for treating or preventing rheumatoid arthritis by using medicinal herb extracts including Curcuma root, Scutellaria baicalensis GEORGE, Achyranthes japonica Nakai, leaf of Eriobotrya japonica Lindley, Acanthopanax senticosus, Taraxacum platycarpum H. Dahlstedt, red ginseng and/or Zizyphus jujuba var. spinosa having activity to inhibit the proliferation of synoviocyte, transcription activity of inflammatory transcription factor (NF-kB), inflammatory cytokines and the creation of nitrogen monoxide.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating rheumatoid arthritis,

The present invention relates to a composition for preventing or treating rheumatoid arthritis using an extract of a medicinal herb. More specifically, the present invention relates to a composition for preventing or treating rheumatoid arthritis, which comprises extracts of eight herbal medicines selected from the group consisting of herbaceous, golden, The present invention relates to a composition for preventing or treating rheumatoid arthritis.

Rheumatoid arthritis is a chronic inflammatory disease characterized by synovial hyperplasia of the joints and destruction of the bones or cartilage. Initially, inflammation occurs in the soft synovial, which mainly surrounds the flexible joint, but the inflammation spreads to the surrounding cartilage and bone, And it is a disease that can show systemic symptoms such as anemia, dry syndrome, subcutaneous nodule, pulmonary fibrosis, vasculitis and skin ulcer.

Although the cause of arthritis is not yet clear, research has been continuing. Although the exact cause of arthritis has not yet been elucidated, autoimmunity is known to be the main mechanism of rheumatoid arthritis. Autoimmunity is a phenomenon that attacks the human body rather than the immune system that protects the human body from the outside. Specifically, the lymphocyte that plays an important role in immunity is a phenomenon in which a part of our body is erroneously recognized as a bacterium invading from the outside It says.

Rheumatoid arthritis is caused by lymphocytes attacking the synovial membrane, which is part of our body. Lymphocytes produce cytokines that stimulate various cells of the synovial membrane and cause inflammation in the process. These cytokines are representative of anti-tumor necrosis factor (TNF-α) and interleukin-1 (IL-1), and they destroy bone around joints and joints and cause systemic symptoms such as fatigue, fever, loss of appetite, It causes.

In addition, antitumor necrosis factor (TNF-α) and interleukin-1 (IL-1) stimulate the production of inflammatory cytokines such as IL-6 by stimulating macrophages, fibroblasts, cartilage cells and osteoclasts, 1 induces the expression of COX-2 or iNOS, thereby increasing the production of NO, an inflammatory mediator, indicating symptoms associated with inflammation (Jeong JG et al. 2004 Biochem Biophys Res Commun 324: 3-7, Alvaro-Gracia JM et al., 1991. J Immunol 146: 3365-71, Grabowski PS et al., 1996 J Rheumatol 35: 207-12, Köjima F et al., 2002 J. Rheumatol 29: 1836-42).

If it represents a symptom associated with rheumatoid arthritis it has been known as an important mediator of the RA involved in various biological phenomena, especially _{NF- K B (Nuclear factor- K B} ) and Cyclooxygenase (COX, cyclic oxygenated enzyme). NF- _K B is a transcription factor that regulates gene expression associated with inflammation and is strongly activated when stimulated by TNF-α, IL-1β, LPS, UV light, and oxidative stress (Dejardin E, 2006, Biochemical pharmacology 72 (9): 1161-1179). The COX enzyme synthesizes PGG2 and PGH2 intermediates from arachidonic acid with hydroperoxidase (HOX) activity together with the function of COX, and PGE2, PGF2, PGD2, prostacyclin and thromboxane A₂ thromboxane A2, TxA2). Among the functions of COX, the function of PGH synthase is involved in the pain and inflammation reaction through synthesis of PGE2.

Although it is effective in treating rheumatoid arthritis with non-steroidal anti-inflammatory drugs (NSAIDs) to help improve joint pain and improve symptoms, this medication prevents cartilage loss or disease progression There is no number, and gastrointestinal side effects such as gastroduodenal ulcers may be a problem when used for a long time. Steroid Hormone System The medication that controls the inflammation, the face should be rounded, the weight gain, diabetes, hypertension, etc.

The development of an effective and safe arthritis treatment with the side effects and defects is continuing, and it is very important to develop a medicament having a low side effect because it is necessary to take the medicine for a long time to treat rheumatoid arthritis It is an urgent matter.

It is an object of the present invention to provide a composition for the prevention or treatment of rheumatoid arthritis by inhibiting synovial cell proliferation and reducing inflammatory cytokines and nitric oxide products to prevent or treat rheumatoid arthritis.

Other and further objects of the invention will be set forth hereinafter.

The invention examples and experiments, as identified in the Examples, turmeric golden wooseul, bipayeop, gasiohgapi, pogongyoung, ginseng and / or the medicinal herb extract is activated synovial cell proliferation inhibitory activity, NF- _K B transcription configured sanjoin below (TNF-a, IL-6) production inhibitory activity and COX-2 production inhibitory activity, as well as inhibitory activity against the increase of COX-2 production.

In view of the foregoing, the present invention relates to a composition for preventing and treating rheumatoid arthritis comprising, as an active ingredient, at least one herbal medicine extract selected from the group consisting of ganoderma, golden, wax, non-leafy ginseng, ginseng root, ginseng root, red ginseng and sanjoin.

Among the active ingredients, Ulgum is a medicinal plant of ginger and Curcuma sp. Cultivated in tropical and subtropical areas, mainly in Southeast Asia. There is often used a tuberous root portion of the equine has keokyu on turmeric (Curcuma wenyujin, YH Chen et C.Ling), autumn turmeric (yellow turmeric) (Curcuma longa Linne), spring corn ( Curcuma longa Salisb.), purple wool ( Curcuma zedoaria ), Kwangseongjak ( Gyeoluleum ) ( Curcuma kwansiensis , SG Lee et CF Liang, Curcuma aeruginosa ), rod opening [ Curcuma phaeocaulis Val. (Ginger and Zingiberaceae)] or Curcuma domestica ), and the like, but are not limited thereto.

Another active ingredient, gold, is a medicinal herb derived from the perennial herbaceous Scutellaria baicalensis , which is a herbaceous plant of the dicotyledonous plant, and has been widely used as a medicinal plant for the treatment of various diseases accompanied by inflammation and oxidative stress Has been used. Baicalin, Baicalein, Woogonin, Woogonoside, and Neobaicalein.

Another active ingredient, the wooseul is soemureup (Achyranthes japonica Nakai or Achyranthes bidentata Blume (Amaranthaceae)) is a sun-dried medicinal herb. It has little odor, it is slightly sweet and has a mucinous appearance. Diuretic, and has been known to have efficacy in the blood.

The other active ingredient, non-foliage, is Eriobotrya japonica Lindley (Rosaceae Rosaceae)) is a dried medicinal herb that has no odor and taste, and is a bit cold. The pharmacological effects of chinhae, geodam, the effect of killing the caterpillars is known. Amygdalin, Ursolic acid, and Oleanolic acid.

Another active ingredient, Acanthopanax Senticosus ) is a deciduous broad-leaved shrub belonging to the Araliaceae such as ginseng. It is also called Siberian Ginseng in Russia and the Americas and Europe. It has been found that the acacia tree contains a large amount of acanthoside and various useful components having excellent biological activity. It is widely used for neuralgia, hypertension, nervous breakdown, diabetes and tonic.

Another active ingredient, Poeurogam, is dandelion ( Taraxacum platycarpum H. around the supposed yakjaeryo, around the country using the sentinel of Dahlstedt) or other dongsok plants (Compositae Compositae), often found in Daw. It has been known to be effective against bacillary action, immune function enhancement, bile secretion action, liver function protection action, diuretic action and the like.

Another active ingredient in ginseng Ginseng is noticeable dried steamed red ginseng did not strip the bark (Panax ginseng . Generally, red ginseng is prepared by processing ginseng after boiling for three consecutive years, such as steaming, drying process, etc. During the manufacturing process, the browning reaction is promoted and the color is dark brown.

Another active ingredient, Sanjin, is Zizyphus jujuba ) is a medicinal product made from ripe seeds of trees and has been used for nervousness, insomnia, The Sanjo-in is the one who picks up the fruit in the fall, dips it in water for one day, removes the pulp, breaks the skin, pulls out the servant and is dried in the sunlight.

As used herein, the term "extract" means an extract of a herb medicine, which is an object of extraction, regardless of the extraction method, in water, anhydrous or lower alcohol (methanol, ethanol, propanol, butanol, n-propanol, iso-propanol and n-butanol) , Extracts obtained by extracting with acetone, ethyl acetate, methylene chloride, chloroform, 1,3-butylenecyclohexane, hexane, diethyl ether or a mixed solvent thereof, and extracts obtained by fractionation of the above- It is understood as meaning. As long as it is extracted through the step of immersing the object to be extracted in the extraction solvent, it is understood that any of extraction methods such as cold beating, refluxing, heating, and ultrasonic wave can be applied. Nevertheless, the extract is preferably obtained by extracting the extraction object with water, ethanol or a mixed solvent thereof, and includes a concentrated liquid extract or a solid extract from which the extraction solvent has been removed.

As used herein, the term "active ingredient" alone means an ingredient which exhibits the desired activity or which can exhibit activity together with a carrier which itself is not active.

The composition for preventing or treating rheumatoid arthritis of the present invention may contain the effective ingredient in any amount (effective amount) as long as it exhibits rheumatoid arthritis prevention or therapeutic activity, depending on the purpose of use, formulation, Will be determined within the range of 0.001 wt% to 99.990 wt% based on the total weight of the composition. Herein, "effective amount" refers to the amount of active ingredient capable of inducing an anti-asthmatic effect. Such effective amounts can be determined experimentally within the ordinary skill of those skilled in the art.

The subject to which the composition of the present invention can be applied (prescription) is preferably a mammal and a person, particularly a human.

In a specific embodiment, the composition of the present invention can be identified as a pharmaceutical composition.

The pharmaceutical composition of the present invention may be in a form suitable for oral use (tablets, suspensions, granules, emulsions, capsules, syrups, etc.), parenteral formulations (sterile injectable aqueous or non- Ointments, ointments, solutions, creams, ointments, gels, lotions, patches, and the like).

The term "pharmaceutically acceptable" as used herein means that the application (subject) does not have an adaptive or higher toxicity (sufficiently low toxicity) without inhibiting the activity of the active ingredient.

Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (e.g., corn starch, potato starch and the like), cellulose, derivatives thereof (e.g. sodium carboxymethylcellulose, ethylcellulose, etc.), malt, gelatin, talc, (E.g., peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, etc.), alginic acid, emulsifiers (e.g., TWEENS), wetting agents (For example, sodium lauryl sulfate), a coloring agent, a flavoring agent, a tableting agent, a stabilizer, an antioxidant, a preservative, water, a saline solution and a phosphate buffer solution. The carrier may be selected from one or more of suitable pharmaceutical formulations according to the formulation of the pharmaceutical composition of the present invention.

The excipient may be selected according to the formulation of the pharmaceutical composition of the present invention. For example, when the pharmaceutical composition of the present invention is prepared by an aqueous suspension, suitable excipients include sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose , Sodium alginate, polyvinylpyrrolidone, and the like can be used. As a suitable excipient when prepared by injection, Ringer's solution, isotonic sodium chloride and the like can be used.

The pharmaceutical composition of the present invention may be administered orally or parenterally.

The daily dose of the pharmaceutical composition of the present invention is usually 0.001 to 150 mg / kg body weight, and may be administered once or several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as route of administration, age, sex, weight, and patient's severity of the patient, the dose is limited in any aspect to the scope of the present invention Should not be understood to be.

The composition of the present invention can be identified as a food composition in another specific embodiment.

The food composition of the present invention can be produced as a health supplement food, a special nutrition supplement food, a functional beverage and the like.

The food composition of the present invention may contain sweetening agents, flavoring agents, physiologically active ingredients, minerals and the like in addition to the active ingredients thereof.

Sweetening agents may be used in an amount that sweetens the food in a suitable manner, and may be natural or synthetic. Preferably, natural sweeteners are used. Examples of natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.

Flavors may be used to enhance taste or flavor, both natural and synthetic. Preferably, a natural one is used. When using natural ones, the purpose of nutritional fortification can be performed in addition to the flavor. Examples of natural flavoring agents include those obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or those obtained from green tea leaves, Asiatica, Daegu, Cinnamon, Chrysanthemum leaves and Jasmine. Also, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, banks and the like can be used. The natural flavoring agent may be a liquid concentrate or a solid form of extract. Synthetic flavors may be used depending on the case, and synthetic flavors such as esters, alcohols, aldehydes, terpenes and the like may be used.

Examples of the physiologically active substance include catechins such as catechin, epicatechin, gallocatechin and epigallocatechin, and vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine and riboflavin.

As the mineral, calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium and zinc can be used.

In addition, the food composition of the present invention may contain preservatives, emulsifiers, acidifiers, thickeners and the like as needed in addition to the above sweeteners.

Such preservatives, emulsifiers and the like are preferably added in a very small amount as long as they can attain an application to which they are added. The term " trace amount " means, when expressed numerically, in the range of 0.0005% by weight to about 0.5% by weight based on the total weight of the food composition.

Examples of the preservative which can be used include calcium sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate and EDTA (ethylenediaminetetraacetic acid).

Examples of the emulsifier which can be used include acacia gum, carboxymethyl cellulose, xanthan gum, pectin and the like.

Examples of the acidulant that can be used include acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, and phosphoric acid. Such an acidulant may be added so that the food composition has a proper acidity for the purpose of inhibiting the growth of microorganisms other than the purpose of enhancing the taste.

Agents that may be used include suspending agents, sedimentation agents, gel formers, bulking agents and the like.

In addition, the food composition of the present invention may contain a powder or extract derived from a natural product in order to improve the flavor or palatability and improve the functionality. Examples of the powder include a pregelatinized powder or extract, a soybean powder or extract, a shell powder or extract, Ginseng extract or extract, cucumber juice or extract, leek juice or extract, spinach juice or extract, lotus juice or extract, juice or extract, pine needles or extract, ginseng juice or extract, Extracts, licorice powder or extracts, garlic powder or extracts, powder or extract of purple powder, powder or extract of sinensis, powder or extract of sinensis, powder or extract of ginger, powder or extract of ginger, jujube powder or extract, Powder or extract, dried powder or extract, Extracts, dandelion powder or extract, ginger powder or extract, green tea powder or extract, omija powder or extract, hinoki powder or extract, dirt powder or extract, oak root powder or extract, cinnamon powder or extract, A knol and the like can be exemplified. The meaning of the above extract is as described above in relation to the extract of Jeju Sori. Such an extract may be obtained by mixing the object to be extracted.

Terms that are not specifically defined in this specification are intended to have a Korean national meaning or a meaning commonly used in the art.

As described above, according to the present invention, it is possible to provide a composition for preventing or treating rheumatoid arthritis comprising, as an active ingredient, at least one herbal medicine extract selected from the group consisting of gilt, golden, wilt, non-leaf, have.

The composition for preventing or treating rheumatoid arthritis of the present invention can be commercialized as a medicine or a food.

Fig. 1 shows the results of experiments for inhibiting the synovial cell proliferation of each herb extract (CL: ugum, EL: non-leaf, UA: ursolic acid, SR: golden, baai: baicalein, ZS: acidophilus, AB: , AS: goosapi TH: Poochung).
FIG. 2 is a graph showing the results of experiments for confirming the synergistic cell proliferation inhibitory effect of the herbal medicine mixed extract (N8).
Fig. 3 is a graph showing the activity of inhibiting NF-KB activation in synovial cells of each herbal extract (CL: ugum, EL: nonfoliated, UA: ursolic acid, SR: golden, Bai: baicalein, ZS: , PG: red ginseng, AS: caustic TH: white pig).
FIG. 4 is a graph showing the results of experiments in which NF-KB activation inhibition activity of eight herbal medicine mixed extracts (N8) was confirmed in synovial cells.
Fig. 5 shows the results of experiments in which the inhibitory activity of IL-6 was inhibited by macrophages (CL: ugum, EL: non-leaf, UA: ursolic acid, SR: golden, BaI: baicalein, ZS: AB: Wassle, PG: Red ginseng, AS: Persimmon TH: Pos.
FIG. 6 is a graph showing the results of experiments in which inflammatory factor (IL-6) inhibitory activity of eight herbal mixed extracts (N8) was confirmed by macrophages.
FIG. 7 is a graph showing the inhibitory activity of TNF-.alpha. Of 8 herbal extracts (N8) from macrophages.
FIG. 8 shows the results of experiments in which the effect of reducing the production of nitrogen monoxide by the eight herbal mixed extracts (N8) was confirmed by macrophages.
FIG. 9 is a graph showing the results of an experiment for confirming chondrocyte proliferation promoting activity of each herb extract (CL: ellum, EL: nonfoliated, UA: ursolic acid, SR: golden, Bai: baicalein, ZS: acidophilus, AB: , AS: goosapi TH: Poochung).
FIG. 10 is a graph showing the results of experiments in which chondrocyte proliferation promoting activity of eight herbal mixed extracts (N8) was confirmed.
FIG. 11 shows the results of experiments in which macrophage cells were observed to inhibit COX-2 expression of eight herbal mixed extracts (N8).

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited by these Examples and Experimental Examples.

≪ Example 1 & gt ; Preparation of each herbal extract

A sample of Chinese medicinal herb was prepared, and the prepared sample was added to 3500 ml of 95% ethanol, and the sample was added to 5000 ml of 95% ethanol once at 100 ° C for 2 hours, And the mixture was refluxed twice at 100 ° C for 2 hours. The extract was filtered through a filter, and the filtrate was concentrated at 50 to 70 ° C using a vacuum condenser. The concentrated extract was lyophilized under a vacuum of -80 캜 using a freeze dryer to prepare each herbal extract.

<Example 2> Preparation of turmeric golden wooseul, bipayeop, gasiohgapi, pogongyoung, Chinese medicine extracts, including both red ginseng and sanjoin

Prepare the kinds and amounts of the medicinal herbals shown in [Table 1] below. Prepare the samples in 3500 ml of 95% ethanol, add the samples to 100 ml of 95% ethanol for 2 hours at 100 ℃ for 2 hours Lt; / RTI &gt; twice. The extract was filtered through a filter, and the filtrate was concentrated at 50 to 70 ° C using a vacuum condenser. The concentrated extract was lyophilized under a vacuum of -80 캜 using a freeze dryer to prepare a herbal medicine extract (hereinafter referred to as N8 extract).

<Example 3> Test cell culture

HIG-82 cells were purchased from the American Type Culture Collection (ATCC, Manassas, Va., USA). F-12 cells containing penicillin-streptomycin and 10% fetal bovine serum (FBS) (Ham`s F-12 nutrient mix medium) in a 5% CO 2 incubator. In addition, RAW 264.7 cells, a mouse macrophage cell line, were cultured in RPMI (GIBCO, Invitrogen) and used in the experiment.

< Experimental Example > Rheumatism  Arthritis Prevention and Therapeutic Activity Experiment

Tests were conducted to examine the effect of the N8 extract on the test cells using the test cells cultured in Example 3 with the extract prepared in Example 1 and Example 2. [ Each herbal extract was used in a concentration range of 12.5 ㎍ to 400 ㎍ per 1 ml of 50% DMSO / EtOH. N8, a complex extract, was used at a concentration of 25 ㎍ to 200 ㎍ per 1 ml of 50% DMSO / EtOH. The results are presented in the sense of ± SEM unless otherwise noted. Student's t-test analysis was used for the statistical significance between the experimental groups. The statistical difference was assessed when the t-test p value <0.05 was considered significant.

<Experimental Example 1> Evaluation of efficacy of herbal extracts in test cell proliferation

IL-1β (5 ng / ml) (Sigma-Aldrich, Saint Louis, Missouri, USA) was added to 96-well microplates (Corning, NY, USA) USA) or cultured without IL-1β.

Celecoxib (Sigma-Aldrich) was used as the control drug. The cultured cells were treated with either of the herbal extracts prepared in Example 1 or the N8 extract and Celcoxib prepared in Example 2, cultured for 2 days or the next day, and cultured for 1 day. Cell proliferation was induced by treatment with 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2Htetrazolium (MTS) (Promega, Madison, After incubation for a period of time, absorbance was measured at 490 nm with ELISA, microplate reader (Power Wavex 340, NIO-TEK-INS TRUMENTS, INC). The proliferation was calculated as a percentage (%) in cells that were not treated with anything, and the results are shown in [Figure 1] and [Figure 2].

Referring to the results of FIG. 1, it can be confirmed that when the herbal medicine, non-flax leaf, golden, tsunami, astringent hair, and porcine root of each herbal medicine of Example 1 were treated, the proliferation of HIG-82 cells was reduced.

FIG. 2a is a graph showing the ratio of HIG-82 cell uptake when two days of HIG-82 cells treated with the N8 extract prepared in Example 2 were cultured. FIG. FIG. 2 is a graph showing the ratio of HIG-82 cell uptake when cultured HIG-82 cells treated with N8 extract prepared at day after cell culture. FIG.

2A, when the N8 extract of Example 2 was used at a concentration of 100 to 200 ug / ml, the HIG-82 cell growth rate was reduced by about 40%. In particular, The N8 extract of Example 2 showed higher inhibitory effect than celecoxib, which is used as an arthritis drug, when the concentration of N8 extract was used at this concentration. Referring to the results of FIG. 2B, And significantly inhibited cell proliferation. In particular, the effect of inhibiting the growth of synovial cells was inhibited by N8 extract in a concentration-dependent manner at a dose of about 200 μg / ml. Thus, it can be seen that the herbal medicine extract of the present invention inhibits HIG-82 synovial cell proliferation.

<Experimental Example 2> Evaluation of efficacy of herbal extracts on NF - _K B transcriptional activation induced by IL- 1β in HIG- 82 cells

One-hundred and ^four HIG-82 cells / well were plated on 96-well microplates (Corning) and infected with reporter plasmid pNF- _K B-SEAP one day later using Hily Max (Dojindo, Japan). One day after the infection, the medium was changed to a clean F-12 medium, and the N8 extract of each of the herbal extracts prepared in Example 1 or the extract of Example 2 was pretreated for 1 hour and then treated with IL-1 And cultured for 6 hours.

10 μl of the supernatant of the HIG-82 cells was added to a new 96-well microplate (Corning) containing 100 μl / well of a SEAP substrate Quanti-Blue (Invivogen), incubated in a 37 ° C. incubator for 1 hour, Absorbance was measured at 620 nm with a Power Wavex 340, NIO-TEK-INS TRUMENTS, INC. The results are shown in FIG. 3 and FIG. 4.

Referring to the results of FIG. 3, it can be confirmed that NF- _K B transcription is activated when the extracts of Korean traditional medicinal herbs of Example 1 are treated with the extracts of Ulgi, Non-leaf, Golden, Sanjôin, Ganoderma and Pseudomonas aeruginosa.

Also, referring to the results of FIG. 4, it can be seen that the N8 extract prepared in Example 2 decreased the increase in NF- _K B transcriptional activation at all concentrations from 25 μg / ml to 200 μg / ml have. Therefore, it can be seen that the herbal medicine extract of the present invention reduces NF- _K B transcriptional activity.

EXPERIMENTAL EXAMPLE 3 Evaluation of the efficacy of herbal extracts on the products of LPS -induced nitric oxide and inflammatory cytokines ( IL- 6 and TNF- alpha) in RAW 264.7 cells

RAW 264.7 cells (1 × 10 ⁵ cells / well) were placed on a 48-well plate (Corning) and the cells were treated with either the herbal extracts prepared in Example 1 or the N8 extract prepared in Example 2 and celecoxib Pretreatment.

To confirm the amount of IL-6 and TNF- [alpha] cytokine produced, the results of measurement in the supernatant of treated RAW 264.7 cells according to the sandwich ELISA kit (Biolegend, USA) 7].

FIG. 5 shows that the extracts of Korean traditional medicinal herbs produced in Example 1 decreased the production of IL-6 in RAW 264.7 cells by the extracts of Ulgi, Non-leaf, Golden, Woose, Red ginseng, Can be confirmed.

Referring to FIG. 6, it can be seen that the N8 extract, which is a mixed herbal medicine prepared in Example 2, decreased IL-6 production in RAW 264.7 cells in a concentration-dependent manner. Also, referring to FIG. 7, it can be confirmed that the N8 extract prepared in Example 2 decreases the production of TNF-? In RAW 264.7 cells in a concentration-dependent manner.

RAW 264.7 cells were treated with LPS (200 ng / ml) (Sigma) for 18-20 hours. Griess Reagent (0.1% N- (1-naphthyl) -ethylendiaminedihydrochoride and 1% Sulfanilamide diluted with 2.5% H3PO4) was added to the supernatant of the treated RAW 264.7 cells to determine the amount of nitric oxide produced. Min, absorbance was measured at 570 nm. The results are shown in Fig.

Referring to FIG. 8, it can be seen that the N8 extract prepared in Example 2 decreased nitrogen monoxide production in RAW 264.7 cells in a concentration-dependent manner.

<Experimental Example 4> Evaluation of efficacy of herbal composition composition for chondrocyte proliferation

The chondrocyte SW1353 cell line cultured in Example 3 was cultured in a 96-well microplate (Corning, NY, USA) at 1 × 10 4 cells / well.

Celecoxib (Sigma-Aldrich) was used as the control drug. The cultured cells were treated with any one of the herbal extracts prepared in Example 1 or the N8 extract and Celcoxib prepared in Example 2 and cultured for 1 day. Cell proliferation was induced by treatment with 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2Htetrazolium (MTS) (Promega, Madison, After incubation for a period of time, absorbance was measured at 490 nm with ELISA, microplate reader (Power Wavex 340, NIO-TEK-INS TRUMENTS, INC). The proliferation was calculated as a percentage (%) in cells that were not treated with anything, and the results are shown in FIG. 9 and FIG. 10.

9, it was confirmed that the extracts of non-foliar, gold, acidophilus, ascidian, red ginseng, goethiopia and porcine subsp. Of each herbal extract prepared in Example 1 showed a tendency to induce the proliferation of SW1353 cells .

Also, referring to the results of FIG. 10, it can be confirmed that when the N8 extract prepared in Example 2 was treated, the proliferation of SW1353 cells, chondrocytes, was induced.

<Experiment 5> The LPS is active Evaluation of efficacy of herbal composition composition for suppression of COX- 2 expression in RAW 264.7 cells

RAW 264.7 cells (1 × 10 ⁶ cells / well) were cultured overnight in a 6-well plate (Corning). The RAW 264.7 cells were pretreated with gold extract or the N8 extract and celecoxib prepared in Example 2 for 2 hours and treated with LPS (200 ng / ml) for 18 hours in each of the herbal extracts prepared in Example 1 Lt; / RTI &gt;

The COX protein was identified by Western blot analysis using an antibody specific for COX-2 (Cell signaling) and Immobilon Western kit (Milipore, USA). The results are shown in FIG. -2 protein was analyzed by LAS-3000 and is shown in Table 2 using AU / mm 2 units.

lane 1 lane 2 lane 3 lane 4 lane 5 lane 6 COX-2 491494.4 4370432 2633184 1259661 2782776 3563781 beta-actin 6953547.25 6479358.85 5816055.13 3018717.32 4018225.2 4621924.12 C / b (ratio) 0.070683 0.674516 0.452744 0.417283 0.692539 0.77106

Referring to the results of FIG. 11 and Table 2, it can be confirmed that the treatment with the herbal medicine extract N8 prepared in Example 2 reduces COX-2 expression in RAW 264.7 cells activated with LPS .

Claims (5)

It contains as an active ingredient, a mixed extract of ginseng, golden, wooze, non-leaf, goose root, ginseng, red ginseng and sanjoin,
The above-mentioned mixed extract was subjected to reflux extraction at a temperature of 100 ° C by adding an aqueous 95% ethanol solution to a mixture of the same weight ratios of Korean wool, golden, wooze, non-leafy, caustic potato, porcine, red ginseng and sanjoin, and the filtrate was filtered, Which is obtained by lyophilization.
delete It contains as an active ingredient, a mixed extract of ginseng, golden, wooze, non-leaf, goose root, ginseng, red ginseng and sanjoin,
The above-mentioned mixed extract was subjected to reflux extraction at a temperature of 100 ° C by adding an aqueous 95% ethanol solution to a mixture of the same weight ratios of Korean wool, golden, wooze, non-leafy, caustic potato, porcine, red ginseng and sanjoin, and the filtrate was filtered, A pharmaceutical composition for preventing or treating rheumatoid arthritis, which is obtained by freeze-drying. delete delete

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