KR20150087552A - Composition for treating and preventing rheumatoid arthritis - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of rheumatoid arthritis. The composition contains an Zizyphus jujube extract and/or a red ginseng extract as an active ingredient. The composition has the synovial cell proliferation suppressing activity and cartilage cell proliferation inducing activity. The composition can suppress the generation of IL-6 and TNF-α which are the main major cytokines relevant to the rheumatoid arthritis and suppress the expression of MMP-2 associated with destruction of the cartilage tissue.

Description

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

The present invention relates to a composition for the prevention or treatment of rheumatoid arthritis, and more particularly to a composition for preventing or treating rheumatoid arthritis comprising an acidolytic extract and / or a red ginseng extract as an active ingredient.

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 preventing or treating rheumatoid arthritis, which comprises an extract of Sanjayin and / or red ginseng as an active ingredient.

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

The present invention relates to a composition for the prevention or treatment of rheumatoid arthritis, which comprises an acidolytic extract and / or a red ginseng extract as an active ingredient, as confirmed in the following Examples and Experimental Examples. As shown in the following examples, the present inventors have found that red ginseng extract and acidosis extract have an activity of inhibiting the proliferation of synovial cells, which is one of the important causes of rheumatoid arthritis. -KB expression was decreased. In addition, the red ginseng extract and the sanjoin extract showed the chondrocyte proliferative activity useful for the treatment of rheumatoid arthritis and the MMP-2 which inhibits the production of IL-6 and TNF-α, the major cytokines associated with rheumatoid arthritis, Lt; RTI ID = 0.0 > expression < / RTI >

Among the active ingredients, the acidophilus was 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.

Another active ingredient, red ginseng, is the ginseng that is not peeled off and steamed to produce dried red ginseng ( 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.

As used herein, the term "extract" is intended to mean any extract or extract of red ginseng or acidophilus to be extracted in water, anhydrous or hydrolysed lower alcohol (methanol, ethanol, propanol, butanol, n-propanol, iso-propanol and n-butanol Etc.), an extract obtained by extracting with acetone, ethyl acetate, methylene chloride, chloroform, 1,3-butylenecrylic, hexane, diethyl ether or a mixed solvent thereof and an extract obtained by fractionation of the above- And is understood to include the 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, which comprises red ginseng extract and / or acidosis extract as an active ingredient.

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 confirming the synergistic cell proliferation inhibitory activity of red ginseng extract and sanjoin extract. FIG. 1A shows the results of treatment of red ginseng extract, and FIG. 1B shows the results of treatment with the acidophilus extract.
FIG. 2 shows the results of experiments in which synovial cells showed inhibitory activity against NF-KB activation of red ginseng extract and acidophilus extract. FIG. 2 (a) is a result of treating red ginseng extract, and FIG. 2 (b) is a result of treatment with a crude acid extract.
FIG. 3 is a graph showing the results of an experiment for confirming chondrocyte proliferation promoting activity of red ginseng extract and sanjito extract. FIGS. 3A and 3B show the results of treatment with red ginseng extract or acidolytic extract, respectively, and FIG. 3C show results obtained by using a mixture of red ginseng and sanjito extract.
FIG. 4 is a graph showing the inhibitory activity of red ginseng extract and acidolytic extract against macrophages. FIGS. 4A and 4B are the results of examining the amount of TNF-α produced after treatment with red ginseng extract or acidophilus extract, respectively, and FIG. 4C is a result of confirming the amount of TNF-α produced after treating a mixture of red ginseng and sanjito extract.
FIG. 5 is a graph showing the results of an experiment in which inflammatory factor inhibitory activity of red ginseng extract and acidophilus extract was confirmed by macrophages. FIGS. 4A and 4B show the results of confirming the amount of IL-6 produced after treating the red ginseng extract or the acidogenic extract, respectively. FIG. 4C shows the result of confirming the amount of IL-6 produced after the treatment of the mixture of red ginseng and sanjito extract.
FIG. 6 is a graph showing the inhibitory activity of red ginseng extract and acidophilus extract on macrophage expression of COX-2 (PG: red ginseng extract, ZS: acidophilus extract).

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.

EXAMPLES Preparation of red ginseng extract and acidolytic extract

200 g of red ginseng or sanjoin was placed in 5000 ml of 94% ethanol and subjected to reflux extraction once at 100 ° C for 2 hours. The extract mixture was filtered through a filter, and the filtrate was concentrated at 50 to 70 ° C using a vacuum decompression condenser. The concentrated extract was lyophilized under a reduced pressure of -80 ° C using a freeze dryer to prepare each herbal extract.

< Experimental Example > Rheumatism  Arthritis Prevention and Therapeutic Activity Experiment

<Experimental Example 1> Experiment on inhibition of synovial cell proliferation

HIG-82 cells (American Type Culture Collection, Manassas, VA, USA), a rabbit knee synovial cell line, was inoculated with F-12 containing penicillin-streptomycin and 10% fatal bovine serum (GIBCO, Invitrogen) (GIBCO, Invitrogen, Carlsbad, Calif., USA) in a 5% CO ₂ incubator.

The cultured HIG-82 cells were incubated with IL-1β (5 ng / ml) (Sigma-Aldrich, St. Louis, Missouri, USA) at 2 × 10 4 cells / well in 96-well microplates Lt; / RTI &gt;

Celecoxib (Sigma-Aldrich) was used as the control drug. The cultured cells were treated with the sample prepared in the examples or Celcoxib and cultured for 2 days. Cell proliferation was treated 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 results are shown in FIG. 1a and FIG. 1b as a percentage of the group treated with IL-1? Alone.

1A and 1B, some treatment groups did not show any significant effect, but generally red ginseng extract and sanjito extract showed anti-proliferative activity of synovial cells.

Experimental Example 2: Synovial cells NF - _K B activation inhibition experiment

SEAP reporter plasmids were transfected overnight in 96-well plates and incubated overnight at 37 ° C in a 5% CO2 incubator. The next day, the drug is treated for each concentration, incubated for 1 hour, treated with IL-1β in the drug-treated wells, and incubated for 6 hours at 37 ° C in a 5% CO2 incubator. After 6 hours, 100 μl of SEAP substrate was added to each well of a new 96-well plate, and 10 μl of the cultured medium was added for 6 hours. After incubation at 37 ° C for 1 hour, the absorbance was measured at 620 nm Respectively.

The results of treating the red ginseng extract and the sanjoin extract are shown in [Figure 2a] and [Figure 2b], respectively. Celecoxib (Sigma-Aldrich) was used as the control drug.

Referring to FIGS. 2A and 2B, it can be seen that the activity of the acidophilus inhibits NF- _K B reporter in a concentration-dependent manner.

<Experimental Example 3> Cholinocyte proliferation promoting activity experiment

Chondrocyte SW1353 cell line (0, 7x10 ⁴ cells / well) was plated on a 96-well plate and cultured for 2 days after drug treatment on the following day, and absorbance was measured at 490 nm by MTS treatment.

The results are shown in Fig. [Fig. 3] shows that both Sanjoin and red ginseng extract promoted the proliferation of chondrocytes in a concentration-dependent manner.

Here, [Fig. 3c] is a result of using a mixture of red ginseng and acidolyzane equivalent as a sample [Fig. 3c].

<Experimental Example 4> Inhibitory activity of inflammatory factors in macrophages

RAW 264.7 cells (1 × 10 ⁵ cells / well) were plated in 48 well plates (Corning) and pretreated with the extract prepared in Example 1 or celecoxib for 2 hours.

 RAW 264.7 cells were treated with LPS (200 ng / ml) (Sigma) for 18-20 hours. To determine the amount of TNF- [alpha] and IL-6 cytokines produced, the supernatants of treated RAW 264.7 cells were measured by the manufacturer's experimental method of a sandwich ELISA kit (Biolegend, USA). Absorbance was measured at 450 nm with an ELISA microplate reader (Power Wavex 340, NIOTEK-INS TRUMENTS, INC). The result of measurement of TNF-α is shown in FIG. 4, and the result of measurement of IL-6 secretion amount is shown in FIG.

As shown in [Figure 4a], it was confirmed that the red ginseng extract prepared in Example 1 reduced TNF- [alpha] production in RAW 264.7 cells [Figure 4b], confirming that the acidogenic extract decreased TNF- [alpha] production I could. In addition, as shown in [Fig. 4c], it was confirmed that TNF-α production was also reduced when a mixture of red ginseng and sanjoin was treated.

As shown in [Fig. 5a] and [Fig. 5b], it was confirmed that IL-6 production was reduced in RAW 264.7 cells in a concentration-dependent manner by the red ginseng extract and the Ranunculin extract. In addition, as shown in [Fig. 5c], the reduction effect of IL-6 production by the mixture of red ginseng and sanjoin was also confirmed.

Experimental Example 5 Expression of COX- 2 in Macrophages

RAW 264.7 cells (1 × 10 ⁶ cells / well) were cultured overnight in a 6-well plate (Corning). The RAW 264.7 cells were treated with 200 ug / ml of the red ginseng extract and / or the acidolytic extract prepared in Example 1, or celecoxib was pretreated for 2 hours and cultured for 18 hours with LPS (200 ng / ml). The COX protein was identified by Western blot analysis using an antibody specific for COX-2 (Cell signaling) and Immobilon Western kit (Milipore, USA) (FIG. 6).

As shown in Fig. 6, the expression of COX-2 was found to decrease when RAW 264.7 cells activated with LPS, especially the acidogenic extract, were treated.

Experimental Example 6 MMP inhibitory activity in macrophages

In a 6-well plate (Corning), 1 × 10 ⁶ cells / well of macrophage RAW 264.7 cells were cultured overnight. The RAW 264.7 cells were treated with 200 ug / ml of the red ginseng extract and / or the acidolytic extract prepared in Example 1, or celecoxib was pretreated for 2 hours and cultured for 18 hours with LPS (200 ng / ml). The matrix metalloproteinase (MMP) protein was identified by Western blot analysis using an antibody specific for MMP-2 (Santa Cruz) and Immobilon Western kit (Milipore, USA). The relative amounts of MMP-2 protein were analyzed by LAS-3000 and are shown in Table 1 as AU / mm &lt; 2 &gt;.

As shown in [Table 1], when the acidogenic extract was treated or the mixture of red ginseng extract and sanjoin extract was treated, the expression of MMP-2 was decreased.

Experimental group Fold increase (MMP / actin) One Negative control group 1.00 2 Positive control (LPS treated group) 1.21 3 LPS + celecoxib 1.16 4 LPS + red ginseng extract 1.22 5 LPS + Sanjo extract 0.79 * 6 LPS + red ginseng extract + sanjoin extract 0.63 *

(* P < 0.05)

Claims (5)

Wherein the composition comprises at least one extract selected from the group consisting of red ginseng extract and acidolytic extract as an active ingredient.
The method according to claim 1,
The composition for preventing or treating rheumatoid arthritis, wherein the effective ingredient includes both red ginseng extract and acidosis extract.
The method according to claim 1,
The composition for preventing or treating rheumatoid arthritis, wherein the extract is obtained by using water, ethanol or a mixed solvent thereof.
4. The method according to any one of claims 1 to 3,
A composition for preventing or treating rheumatoid arthritis, wherein the composition is a pharmaceutical composition.
4. The method according to any one of claims 1 to 3,
A composition for preventing or treating rheumatoid arthritis, wherein the composition is a food composition.

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