KR101285234B1 - Pharmaceutical Compositions for Preventing or Treating Arthritis Comprising Cynanchum Atratum Extracts - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising a white rice extract (Cynanchum atratum) comprising a steroidal glucoside compound exhibiting potent anti-inflammatory activity against arthritis and the white rice extract having an anti-inflammatory effect.
The extract or fraction of Cynanchum atratum of the present invention is a herbal derived from natural products, has no toxicity and no side effects, and significantly inhibits the production of nitric oxide and tumor necrosis factor-α, which are key mediators of the inflammatory response, and inflammation. Since it has an excellent anti-inflammatory effect, such as a mediator PGE ₂ inhibitory action, the composition containing it can be useful for fundamentally treating and preventing arthritis inflammatory diseases, as well as alleviating and improving symptoms.

Description

Pharmaceutical Compositions for Preventing or Treating Arthritis Comprising Cynanchum Atratum Extracts}

The present invention relates to a pharmaceutical composition comprising a white rice extract (Cynanchum atratum) comprising a steroidal glucoside compound exhibiting potent anti-inflammatory activity against arthritis and the white rice extract having an anti-inflammatory effect. More specifically, the present invention relates to an arthritis therapeutic composition having a inflammatory disease and analgesic inhibitory effect and cartilage protection effect of a joint by extracting and concentrating a bioactive component of Cinnanchum atratum, and a method of manufacturing the same.

An inflammatory response is a mechanism for repairing and repairing an injured area when an invasion that causes any organic change in the body or tissue, such as physical action, chemicals, or bacterial infection, is applied. Once irritated, topical histamine, serotonin, bradykinin, prostaglandins, hydroxyl-eicosatetraenoic acid (HETE) Vascularly active substances, such as leukotriene, are liberated, increasing inflammation and causing vascular permeability. Appropriate inflammation that occurs early in the injury is a normal response to bring about a quick repair to the site of injury.

However, prolonged inflammatory reactions can make recovery more difficult by repairing damaged areas and damaging surrounding cells, reducing their defenses to surrounding tissues. In addition, prolonged inflammatory reactions can lead to persistent damage to surrounding tissues, resulting in some cancers.

Therefore, tissues with inflammatory reactions should be treated with appropriate anti-inflammatory agents in order for the repair program to work quickly. However, steroidal agents used to treat inflammatory diseases have many side effects and limit their use. Therefore, the development of anti-inflammatory agents with excellent anti-inflammatory effects and fewer side effects is required.

Arthritis-related disease is a representative degenerative and refractory disease in which about 12% of the world suffers. There are more than 2 million patients in Korea. Arthritis is a general name that causes inflammatory changes in the musculoskeletal and connective tissues of the body, resulting in systemic symptoms in the musculoskeletal system. The disease is characterized by chronic inflammation that affects the joints, bones, cartilage tissue or spinal cord, often causing permanent tissue damage, malformations, degeneration and disorders. (Hofbause, L.C., et al., Arthritis and Rheumatisu 44: 253-259, 2001). Arthritis is classified as degenerative arthritis (osteoarthritis), rheumatoid arthritis, extra-articular rheumatoid or collagen disease.

The most common degenerative arthritis among arthritis diseases is a disease in which local degenerative changes occur as the articular cartilage wears off. Degenerative arthritis causes degeneration such as aging in chondrocytes constituting the joint, which inhibits the synthesis of type-II collagen and proteoglycan, which are the matrix substances of the joint, and at the same time, interleukin. Matrix metalloproteinases that degrade joint substrates by the production of inflammatory cytokines such as -1β (interleukin-1β, IL-1β) and tumor necrosis factor-α (TNF-α) Metalloproteinase (MMP) is a disease caused by the destruction of joint tissue due to increased synthesis and activity in the joint cells. In addition, arthritis is exacerbated by the production of nitric oxide (NO) by inflammatory cytokines and the production of self-amplifying cytokines by nitric oxide produced, which leads to the synthesis of more MMPs, thereby facilitating the decomposition of the articular matrix. At the same time, inflammatory cytokines increase the production of the lipid metabolite, prostaglandin E2 (PGE2), which induces an inflammatory response in arthritis. The cause is uncertain, but it is known that the disease is deeply related to aging or excessive weight, in which the degenerative change of articular cartilage is the first. Degenerative changes begin in articular cartilage leading to necrosis of cartilage cells, and the substrate is destroyed by cathepsin B, catepsin D, collagenase, and the like. The production of proteoglycan and collagen does not keep up with the degree of destruction, and the ability of cartilage to adapt to external forces gradually decreases, resulting in fine fractures in the cartilage of the cartilage. As the disease progresses, hardening of the subchondral bone, hyperplasia of the bone around the joint, deformation of the joint, and the like, the surface of the cartilage becomes rough and the inflammatory response is repeated in the joint cavity wrapped in the joint membrane. As a result, repetitive pain, joint stiffness, gradual movement disorder of the joint, and the like are caused.

Among arthritis, rheumatoid arthritis is a systemic and chronic inflammatory disease in which pathological symptoms are mainly symmetrically on movable joints, and it is known as an autoimmune disease whose cause is caused by an abnormality of the immune system, and the cause is not known yet. The disease is characterized by persistent inflammatory synovitis causing cartilage destruction and bone erosion, causing structural malformations of the surrounding joints. Symptoms associated with rheumatoid arthritis include joint edema, joint tenderness, inflammation, early stiffness and especially pain when bending the joint. Subjects in the advanced stage of arthritis develop bone damage and structural damage, including joint destruction (Firestein, G.S., Nature 423: 356-361, 2003). In addition, the patient may have other clinical symptoms such as damage to various organs, including damage to the skin, kidneys, heart, lungs, central nervous system and eyes due to vasculitis associated with the autoimmune process.

Other symptoms associated with arthritis include increased erythrocyte sedimentation rate and increased serum C-reactive protein (CRP) and / or soluble IL-2 receptor (IL-2r) concentrations. Erythrocyte sedimentation rate is increased in almost all patients with active rheumatoid arthritis. The concentration of serum C-reactive protein is also increased, which is related to disease activity and the possibility of progressive joint damage. In addition, the concentration of soluble IL-2r, a product of T-cell activation, in serum and synovial fluid of patients with active rheumatoid arthritis is also increased (Udagawa, N., et.al., Arthritis and Research 4: 281-289). , 2002).

Th1 type CD4 + T cells are known to play an important role in the development and persistence of rheumatoid arthritis. In other words, CD4 + T lymphocytes stimulate macrophages and synovial cells through signal transduction by soluble substances such as interferon-gamma (IFN-γ) and IL-17 and cell surface substances such as CD69 to stimulate inflammatory cytokines (TNF-α, IFN). -γ, GM-CSF, IL-2, IL-6) and matrix metalloproteinase (MMP). These secreted cytokines promote the growth of the synovial membrane to form pannus, which together with MMP cause cartilage destruction. In addition, activated CD4 + T cells activate B cells through CD40L, CD28, a1b2 integrin-mediated cell surface contact to produce antibodies including rheumatoid factors. As activated, CD4 + T cells express osteoprotegerin ligand (OPGL) in vyas to promote osteoclastogenesis and play a crucial role in bone destruction (Kong YY, et al., Nature 402). : 304-309, 1999). Activated macrophages, fibroblasts, etc. secrete substances such as VEGF and FGF to promote new blood vessel formation. Activated vascular endothelial cells in synovial tissue secrete chemokines, such as IL-8, induce expression of adhesion molecules, and promote infiltration of inflammatory cells to achieve an amplification cycle of the inflammatory response. Rheumatoid arthritis is also thought to be a T-cell-mediated autoimmune disease involving antigen-nonspecific cell-cell interactions between T-lymphocytes and antigen presenting cells. In general, the magnitude of the T-cell response is determined by a co-stimulatory response induced by the interaction between the T-cell surface molecule and its ligand. The main co-stimulatory signal is the interaction between the T-cell surface receptors CD28 and CTLA4 and their ligands, eg, the B7-related molecules CD80 (ie B7-1) and CD86 (ie B7-2) on antigen presenting cells. (Linsley, P., et al., Ann. Rev. Immunol. 11: 191-212, 1993). T-cell activation in the absence of costimulation results in an anergic T-cell response in which the immune system does not respond to stimuli (Schwartz, RH, et.al., Cell 71: 1065-1068, 1992) .

When osteoarthritis is induced, pain increases with an inflammatory response in the joint, because the pain increases the production of prostaglandin E2 (PGE2), an important factor associated with pain induction in the synovial fluid. Cyclooxygenase-2 (COX-2) is an enzyme that synthesizes prostaglandin E2, a pain-causing substance, which is an inflammatory cytokine such as NO (nitric oxide), TNF-α and IL-1β. , TNF-α), chemokines, proteases and the like are associated with inflammation. These inflammatory reactions are also observed in the early stages of osteoarthritis. Inflammation of the synovial membrane can lead to elevated serum hyaluronic acid (HA) levels. Sensory nerve fibers are distributed in the synovial membrane, and IL-1β and TNF-α can stimulate or be sensitive to nociceptors, which are receptors that detect or damage harmful organisms distributed in the synovial membrane. Can be. These inflammatory cytokines release PGE2 and histamine from chondrocytes and mast cells, which in turn stimulate painful receptors. In osteoarthritis, the cartilage is destroyed along with the inflammatory response and proteoglycan is released into the synovial fluid, increasing the amount of synovial fluid and the concentration of proteoglycan in the synovial fluid. Proteoglycan is composed of protein and sugar, and is a complex molecule that gives elasticity to cartilage to allow the body to bend and squeeze by forming a densely packed structure or framework in the cartilage, which is entangled with collagen fibers. The proteoglycan also functions as a sponge that can contain water in the cartilage tissue so that the cartilage can continue to exercise joints. Cartilage tissue in which osteoarthritis occurs is destroyed by matrix metalloproteinase (MMP), and as osteoarthritis progresses, expression and activity of MMP-1, -2, -3, -8, -9, and -13 increase (Wieland, HA). , et. al., Nat Rev Drug Discov 4: 331-344, 2005).

Currently used treatment methods for degenerative arthritis and rheumatoid arthritis are as follows. Drugs commonly used in early treatment are nonsteroidal anti-inflammatory drugs (NSAIDs), which may slightly improve symptoms but do not prevent joint cartilage loss or disease progression. Over the long term, severe side effects occur in the gastrointestinal, central nervous system, hematopoietic organs, kidneys, liver, etc., and about half of the patients using this therapy must stop treatment within one year (Langenegger, T., et. al., Clin Orthop. 366: 22-30, 1999). The next step is the drug modifying anti-rheumatic drugs such as gold drugs (gold sodium thimalate, gold sodium thiosulfate), penicillamine, anti-malarials, DMARDs) and the like. However, these agents also slightly reduce the progression of arthritis, but can have serious side effects, so only 5-15% of patients will be treated after 5 years of treatment with Disease Modifying Anti-Rheumatic Drug (DMARD). I keep using this drug. If the above treatments are no longer effective, rheumatoid joints may need to be replaced with artificial joints (Simon, LS, Int. J. clin. Pract., 54: 243-). 249, 2000).

Nitric oxide (NO) is produced in endothelial cells, neutrophils, etc., and is also produced when L-arginine is converted to citrulline by nitric oxide synthase (NOS). In addition, nitric oxide acts as an intercellular messenger in most organs and acts as a homeostasis of blood vessels, inhibits leukocyte or platelet adhesion to vascular endothelial cells, inhibits platelet aggregation and neurotransmitters. In addition, the immune system is involved in macrophage and antimicrobial defenses, and reacts easily with superoxide anions to produce peroxynitrite (ONOO-), a highly reactive oxidant. NO causes neurodegenerative diseases, chronic inflammation, sepsis, high blood pressure, thrombosis, renal failure, ARDS (adult respiratory distress syndrome), brain disorders caused by AIDS, bronchial spasms, seizures, and male erectile dysfunction. Nitric oxide (NO) is another type of active species that is highly cytotoxic but plays an important role in intracellular or intracellular signaling and is an immune response as an Endothelium Derived Relaxing Factor (EDRF) in vivo. Is of great interest because of its potential as a degenerative neurotransmitter, particularly in memory and in maintaining cell homeostasis. Nitric oxide is synthesized from L-arginine by nitric oxide synthase (NOS) in several tissues.NOS has three isoforms, type I (neuronal nitric oxide synthase, nNOS) and type II (inducible nitric). oxide synthase (iNOS) and type III (endothelium nitric oxide, ecNOS), type I and III are constitutive forms (cNOS) regulated by Ca ²⁺ and calmodulin, and type II is used in post-transcription. It is referred to as a cytokine-inducible form (iNOS) that is regulated by cNOS, which is present in the cell and maintains normal physiological function. It is expressed after exposure to (cytokine) and produces a large amount of NO, causing indirect effects such as nitrosation and nitration and oxidative reactions. NO produced by activating cells is well known for showing cytotoxicity to surrounding tissues, and since NO has a very short half-life of 6-10 seconds, it is difficult to detect and study directly. Stabilized byproducts such as nitrite and nitrate are measured indirectly.

As the inflammatory response progresses, the expression of inflammatory cytokines continues to increase, which leads to prolonged inflammatory responses. Inhibiting the expression of these inflammatory cytokines IL-1β, IL-6 and IL-8 relieves inflammation. Very important. In addition, the anti-inflammatory effect is shown by inhibiting the expression of NF-κB, a transcriptional regulator that induces the expression of inflammatory cytokines (Inoue et al., Cancer Sci, 98, 268-274, 2007). Toll-like receptors, which are sensitive to bacteria, viruses, or inflammatory inducers and transmit signals into cells, activate cytokines that trigger inflammation by activating transcriptional regulators such as NF-κB and IRF3 through intracellular signaling systems, It promotes the expression of chemokines and the like. (Kollisch et al., Immunology, 114, 531-541, 2005). The search for a substance that inhibits the expression of inflammatory cytokines or inhibits the expression of the transcriptional regulator NF-κB or IRF3 will provide a functional cosmetic or medical agent useful for anti-inflammatory treatment and improvement.

Therefore, the present inventors measured nitric oxide (NO) production, tumor necrosis factor-α, and representative inflammatory cytokines, Interleukin-, in the process of searching for substances that may exhibit anti-inflammatory effects by enhancing immune function. Concentration test method of 1β and confirmation of cytotoxic efficacy were verified the efficacy of non-toxic food ingredients, natural products or conventional herbal medicines. In addition, extracting an excellent anti-inflammatory bioactive component for arthritis from Cynanchum atratum by confirming the analgesic inhibitory effect, the reduction of edema rate, and the anti-inflammatory effect of arthritis through animal experiments while maintaining the cartilage and bone tissue of the arthritis. , Fractions were prepared. Particularly, the present invention was completed by separating extracts showing high immune cell activation effect in the chloroform layer among white rice fractions, having excellent destruction ability of inflammatory cells in vivo, and effectively inhibiting the growth of inflammatory cells and demonstrating its efficacy.

It is an object of the present invention to provide a pharmaceutical composition useful for the treatment and prevention of immune function enhancement or inflammatory diseases containing extracts of bioactive components of Cinnanchum atratum. In order to achieve the above object, the present invention provides an anti-inflammatory pharmaceutical composition and a pharmaceutical composition for the treatment and prevention of inflammatory diseases containing a physiologically active ingredient of the white rice (Cynanchum atratum) showing an immune activity and anti-inflammatory effect as an active ingredient to provide.

In order to achieve the above object, according to a preferred embodiment of the present invention, there is provided a pharmaceutical composition for preventing and treating arthritis, including a steroidal glucoside extracted from white rice (Cynanchum atratum) as an active ingredient.

According to another suitable embodiment of the present invention,

The steroidal glucoside provides a pharmaceutical composition for preventing and treating arthritis, characterized in that the compound represented by the following formula (1).

R is

or

to be.

According to another suitable embodiment of the present invention, the steroidal glucoside is added with 5-20 L of water or alcohol solvent per kg of dry root of white rice and left at 20-50 ° C. for 0.5-2 days, followed by filtration and evaporation under reduced pressure. To obtain an alcohol extract; Adding a solvent in which water and hexane are mixed 1: 1 to the alcohol extract, and then extracting an organic material by adding chloroform to the separated water layer; And extracting the organic material by fractionation with four steps of chromatography.

According to another suitable embodiment of the present invention, the alcohol solvent provides a pharmaceutical composition for preventing and treating arthritis, characterized in that the ethanol or methanol.

According to another suitable embodiment of the present invention, the chromatography is prevention of arthritis, characterized in that one selected from the group consisting of normal phase vacuum flash chromatography, reverse phase open column chromatography and C18 reverse phase semi-preparative high performance liquid chromatography. Provided is a therapeutic pharmaceutical composition.

The extract of Cynanchum atratum of the present invention is Nitric Oxide and inflammation mediated cytokines Tumor necrosis factor-alpha, Interleukin-1 beta, inflammatory mediator prostaglandin E _{2 in} macrophages involved in the inflammatory response Inhibits the activity of (Prostaglandin E ₂ ). The pharmaceutical composition containing the white rice extract of the present invention may be developed as an anti-inflammatory agent and may also help in the prevention of degenerative diseases such as gastritis, colitis, hepatitis, arthritis and arteriosclerosis, and cancer caused by inflammatory reactions.

FIG. 1 shows an HPLC chart of a steroidal glucoside compound which is an index component of Cynanchum atratum extract.
Figure 2 measures the toxicity of the immune cells of the white rice extract of Comparative Example 1, Comparative Example 2 and Example 1.
Figure 3 measures the amount of NO production inhibition of the white rice extract of Comparative Example 1, Comparative Example 2, Example 1.
4 is a measure of the amount of TNF-α inhibiting the production of the white rice extract of Comparative Example 1, Comparative Example 2, Example 1.
5 is a measurement of the amount of IL-1β production inhibition of the white rice extract of Comparative Example 1, Comparative Example 2, Example 1.
Figure 6 measures the amount of inhibition of production of PGE ₂ of the white rice extract of Comparative Example 1, Comparative Example 2, Example 1.
Figure 7 shows the manufacturing process of the white rice extract of Example 1.

White rice extract of the present invention is the step of adding 5 to 20 liters of water or alcohol solvent per kg of dried root of white rice and left at 20 to 50 ℃ for 0.5 hours to 2 days, followed by filtration and evaporation under reduced pressure to obtain an alcohol extract; Adding a solvent in which water and hexane are mixed 1: 1 to the alcohol extract, and then extracting an organic material by adding chloroform to the separated water layer; And extracting the organic material by fractionation with four steps of chromatography.

First, the roots of white rice (Cynanchum atratum) are washed with water to remove heavy metals or contaminants, dried, and then ground. Next, using about 5 to 25 times the dry weight, preferably about 20 times the volume of water, lower alcohols such as methanol, ethanol or a mixed solvent thereof having a mixing ratio of about 1: 0.1 to 1:10 Extract. Preferably extracted with methanol at an extraction temperature of 20 to 100 ° C., preferably at 20 to 50 ° C. for about 0.5 hours to 2 days, preferably 1 hour to 1 day, and the extraction frequency is 1 to 5 times, preferably Preferably two to three consecutive extractions.

Next, the extracted material is filtered under reduced pressure, and the filtrate is concentrated under reduced pressure at 20 to 100 ° C., preferably at 50 to 70 ° C., using a vacuum rotary concentrator, and the extracted residue is dried with a vacuum freeze dryer to obtain an alcohol extract. Each extract obtained was used for the experiment while stored at -20 ℃.

Water and hexane are added to the obtained alcohol extract in a 1: 1 ratio, mixed, and separated into a hexane layer and a water layer. Chloroform is added to the separated water layer, mixed and left to stand. After the water layer and the chloroform layer is separated, the chloroform layer is dried under reduced pressure to prepare a chloroform extract. At this time, the vacuum drying conditions are as described above.

Next, the chloroform extract is successively separated and purified by normal phase vacuum flash chromatography, reverse phase open column chromatography, C18 reverse phase semi-preparative high performance liquid chromatography, and reverse phase analysis HPLC column to prepare a white rice extract of the present invention. In this case, it is preferable to select an effective fraction through the anti-inflammatory efficacy test for each fraction and to separate and purify the selected effective fraction by chromatography in the next step.

White rice extract prepared in the present invention is a steroidal glucoside (steroidal glucoside) of a substance containing a compound represented by the following formula (1) as an active ingredient.

Formula 1

In which R is

or

Or a sugar similar thereto.

In particular, the present invention comprises a glaucogenin C-mono-D-thevetoside represented by the following formula (2) as an active ingredient.

White rice extract of the present invention may also include isomers of the compound.

In order to investigate the efficacy of the anti-inflammatory agent containing the extract of Cinnanchum atratum obtained in the above, the concentration of the extract in RAW 264.7 mouse macrophages and C57BL / 6 mice stimulated with lipopolysaccharide (LPS) The effect of the extract according to the present invention on the production of nitric oxide in macrophages and the inflammatory cytokine TNF-α was determined by molecular biological experiments with biochemical analysis. (Tumor necrosis factor-alpha), IL-1β (Interleukin-1 beta) and pro-tags pro-taglandin E ₂ to inhibit the inflammation was confirmed to be excellent.

When oral administration of the extract of Cynanchum atratum of the present invention obtained by the above method to the rat and induce edema in the hind paw with carrageenan, it showed a significant edema inhibition rate compared to the control, Cinnanchum atratum extract When orally administered to rats and mycobacterium butyricium-containing CFA (Freund's complete adjuvant) induces chronic arthritis in the hind paws, even when compared to the control group it was confirmed that the excellent anti-inflammatory analgesic effect.

In addition, the white rice of the present invention (Cynanchum atratum) is a drug that has been used for a long time as a drug extracts of the present invention extracted therefrom also have no problems such as toxicity and side effects.

Pharmaceutical compositions for the treatment and prevention of anti-inflammatory and inflammatory diseases containing the biologically active component of the white rice (Cynanchum atratum) of the present invention, preferably containing from 0.1 to 50% by weight of the white rice extract relative to the total weight of the composition Do.

In addition, the composition comprising the bioactive component of Cinnanchum atratum of the present invention may further comprise a suitable carrier, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of fractions containing bioactive ingredients that exhibit excellent efficacy in arthritis of the present invention may be used in the form of their acceptable salts and may also be used alone or in combination with other pharmaceutically active compounds, as well as in suitable collections. Can be used.

The pharmaceutical compositions containing the fractions according to the present invention can be administered orally or parenterally in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, And can be used as formulations. Examples of carriers, excipients and diluents that can be included in the composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. 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. High blood preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, or the like in the extract or fraction. (sucrose), lactose (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts or fractions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract or fraction of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

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

The present invention provides a health functional food comprising the extract or fraction and food acceptable food supplements exhibiting a prophylactic effect of inflammatory diseases or arthritis. Foods to which Cynanchum atratum extract or fraction can be added include, for example, various food rates, beverages, gums, teas, vitamin complexes, and health functional foods.

White rice (Cynanchum atratum) extract of the present invention is a drug that can be used with confidence even when taken for a long time for the purpose of prevention because there is little toxicity and side effects.

In addition, the extract or fraction of Cynanchum atratum of the present invention exhibits anti-inflammatory activity in the molecular biological findings of arthritis, and has excellent anti-inflammatory and analgesic activity in inflammatory and pain-inducing animals, and thus a pharmaceutical composition useful for the prevention and treatment of arthritis and It can be used as a dietary supplement.

Hereinafter, the present invention will be described in detail by comparative examples, examples and experimental examples. However, the following Comparative Examples, Examples and Experimental Examples are merely illustrative of the present invention, but the content of the present invention is not limited thereto.

Comparative Example 1: Preparation of White Rice (Cynanchum atratum) Extract

1 g of methanol was added to 200 g (dry weight) of dried roots of Cynanchum atratum purchased from Junghan Intercorp, and left for 48 hours. After filtering, the solvent was evaporated and dried three times under reduced pressure to obtain 40.4 g of methanol extract. Obtained.

Comparative Example 2: Preparation of Cinnanchum atratum Fraction

Water and hexane (each 200 ml) were added and mixed with respect to the extract illustrated in Comparative Example 1, the hexane layer and the water layer were separated, and 200 ml of chloroform was added to the water layer and mixed. Thereafter, the water layer and the chloroform layer were separated, and the chloroform layer was dried under reduced pressure to obtain 8.4 g of an organic substance as a chloroform extract.

Example 1 Preparation of Bioactive Fractions of Cynanchum atratum

1 g of methanol was added to 200 g (dry weight) of dried roots of Cynanchum atratum, and the mixture was left for 48 hours, and then filtered three times. The solvent was evaporated to dryness under reduced pressure to obtain 40.4 g of methanol extract. Water and hexane (each 200 ml) were added to the extract, followed by mixing. The hexane layer and the water layer were separated, and 200 ml of chloroform was added to the water layer and mixed. Thereafter, the water layer and the chloroform layer were separated, and the chloroform layer was dried under reduced pressure to obtain 8.4 g of an organic material. The obtained organic material was fractionated by normal phase vacuum flash chromatography. The column is a glass filter column 100ⅹ95mm (inner diameter ⅹ length), the fixed phase is semi-preparative silica for TLC, 50 to 100% (v / v) dichloromethane, methanol mixed solution as the eluent, washing solution As 100% methanol was used. For each fraction, the amount of NO induced by lipopolysaccharide, an inflammation-inducing factor in RAW 264.7 cells, was measured, and tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-), which are representative inflammatory cytokines, were measured. The amount of 1β) and prostaglandin E ₂ (PGE ₂ ) were measured and an effective fraction of 3.84 g was selected by comparing the positive control group without any lipopolysaccharide with the negative control group with the lipopolysaccharide. The selected active fractions were again fractionated using reversed-phase open column chromatography. At this time, the column was a glass filter column 25ⅹ400mm (inner diameter 고정 length), the stationary phase was C18 semi-preparative silica for TLC, the eluent was used 70% methanol aqueous solution, the washing solution was used 100% methanol. Each fraction was subjected to the same anti-inflammatory effect test as above to select an effective fraction of 0.24 g, and a C18 reversed-phase semi-preparative HPLC column (YMC-ODS column, particles). 5 μm in diameter, 10 X 250 mm (inner diameter X length), eluent: 70% (v / v) methanol, elution rate: 2 ml / min, refractive index detector) to separate and purify the viscous liquid component eluted at a holding time of 27 minutes. Obtained. Again C18 reversed-phase analytical HPLC column (YMC-ODS column, particle diameter 5um, 4.6 X 250mm (inner diameter X length), eluent: 70% (v / v) methanol, elution rate: 0.8ml / Minute, refractive index detector), glucogenin C-mono-D-tebetoside, a steroidal glucoside-based substance that elutes at 14.5 minutes of retention time. thevetoside) was extracted 25.9 mg. The HPLC chart of the extracted material is shown in FIG. 1.

[Experimental Material]

DMEM cultures, Fetal bovine serum (FBS), penicillin and streptomycin were purchased from Life Technologies Inc. (Grand Island, NY). 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt: MTS (a)]

[3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt; MTS (a)], dimethyl sulfoxide (DMSO), and E. Coli lipopolysaccharide (Lipopolysaccharide, LPS) were purchased from Sigma Chemical Co., Mo., USA. The Griries reagent system kit for measuring nitrogen oxides was purchased from Promega (Promega, WI, USA), and the Elisa kit for measuring TNF-α and IL-1β cytokine levels. ELISA kit) was purchased from ebioscience, CA, USA.

Experimental Example 1. Cell Culture

RAW 264.7 mouse macrophage (Macrophage) contains fetal bovine serum (FBS) (10%) and antibiotics (antibiotics-antimycotics, 100U / ml penicillin G sodium, 100ug / ml streptomycin sulfate and 0.25ug / ml amphotericin B) It was passaged every other day at 37 ℃, 5% CO ₂ conditions in DMEM medium containing.

Experimental Example 2. Cytotoxicity Test (MTS assay)

RAW 264.7 mouse macrophage (Macrophage) was dispensed in 96-well plate at 5 x 10 ³ per well incubated for 24 hours at 37 ℃, 5% CO ₂ conditions, Comparative Example White rice extract and celecoxib of 1, Comparative Example 2, and Example 1 were administered at the concentrations of 50 µg / ml, 100 µg / ml, 200 µg / ml, and 500 µg / ml, respectively. After administration again incubated for 48 to 72 hours at 37 ℃, 5% CO ₂ conditions. After incubation, [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt; MTS (a)] solution is added to 20 μl of each well and allowed to react while incubating at 37 ° C., 5% CO ₂ for 1 to 4 hours. After the reaction was stopped, absorbance was measured using an absorbance spectrometer at 490 nm. The positive control group without the test substance was converted into 100% survival rate to calculate the survival rate in the experimental group for the test substance and is shown in FIG. 2.

Referring to Figure 2, the liver cytotoxic effect of the white rice extract of Comparative Example 1, Comparative Example 2 and Example 1 in RAW 264.7 mouse macrophage cells (positive control) without any test substance added Or, compared with celecoxib, it was confirmed that they are safe substances that do not affect cell survival.

Experimental Example 3. Measurement of Nitric Oxide (NO) Formation

RAW 264.7 cells were suspended in DMEM medium containing 10% FBS free of phenol red and placed in 5 × 10 ⁵ cells for each well of a 24-well plate at 37 ° C., 5 Incubated for 24 hours at% CO ₂ culture conditions. 1 µg / ml LPS, white rice extract and celecoxib of Comparative Example 1, Comparative Example 2 and Example 1 were respectively 10 µg / ml, 50 µg / ml, 100 µg / ml and 200 µg / ml in RAW 264.7 cells. Concentration is administered and incubated for 20 hours with simultaneous treatment with DMSO. 100 μl of the supernatant was reacted with 180 μl of Griess reagent (0.1% naphthylethylenediamine solution and 1% sulfanilamide in 5% H ₃ PO ₄ solution) to measure absorbance at 540 nm. It was. The calibration curve was prepared using sodium nitrite solution, and the absorbance average was converted into the amount of nitrite using the solution. Based on the amount of nitrite in the LPS-treated group, the NO ₅₀ inhibitory activity of the test substance treated group was determined by using non-linear regression analysis. % Of the inhibitory concentration) is shown in FIG.

As confirmed in FIG. 3, the production amounts of nitric oxide (NO) were compared when the white rice extracts of Comparative Example 1, Comparative Example 2 and Example 1 were administered to RAW 264.7 mouse macrophage cells. As a result, it was confirmed that Example 1, which is a pure anti-inflammatory active material, showed an excellent inhibitory effect compared to celecoxib, which is an existing inflammatory treatment.

Experimental Example 4 Measurement of TNF-α and IL-1β Cytokine Levels

RAW 264.7 cells were cultured by dispensing 5 × 10 ⁵ cells per well into a 24-well plate, and then 1 μg / ml LPS, Comparative Example 1, Comparative Example 2 and Example 1 White rice extract and celecoxib were administered at concentrations of 10 μg / ml, 50 μg / ml, 100 μg / ml and 200 μg / ml, respectively. After culturing for 5 hours at 37 ° C. and 5% CO ₂ again, cell cultures were taken and TNF-α and IL-1β contents were measured using an enzyme immunoassay kit (EIA) kit. Cell cultures were added to a 96-well plate to which capture antibodies for each cytokine were attached, and after 24 hours reaction, diluted TNF-α, IL-1β antibody and horseradish peroxy Secondary antibodies bound to horseradish peroxidase were each reacted at room temperature for 2 hours. After the reaction, the mixture was washed 5 times with 1 × phosphate buffered saline tween (1 × PBST), and then reacted for 30 minutes by adding the following enzyme substrate. Then 2N-sulfuric acid was added to stop the reaction. Absorbance developed after the reaction was stopped at 450 nm wavelength using an absorbance spectrophotometer. The contents of TNF-α and IL-1β were converted using a standard curve obtained from the reaction of the standard.

As confirmed in Figure 4, when the white rice extract of Comparative Example 1, Comparative Example 2 and Example 1 in RAW 264.7 mouse macrophage cells, the inflammatory cytokine TFN-α (Tumor necrosis factor) As a result of comparing the expression concentration for -α), it was confirmed that Example 1 significantly inhibited TNF-α for each concentration.

As shown in Figure 5, when the white rice extract of Comparative Example 1, Comparative Example 2 and Example 1 in RAW 264.7 mouse macrophage cells, the inflammatory cytokine IL-1β (Interleukin-1β As a result of comparing the expression concentration for), it was confirmed that Example 1 significantly inhibits IL-1β by concentration. It was confirmed that the inhibition of expression of TNF-α and IL-1β in FIGS. 4 and 5 showed an effect corresponding to the positive control group or celecoxib without addition of LPS (Lipopolysaccharide) which causes inflammation.

Experimental Example 5. prostaglandin _{E 2 (Prostaglandin E 2, PGE} 2) measurements generated

RAW 264.7 cells were suspended in DMEM medium containing 10% FBS and 1 × 10 ⁵ cells were added to each well of a 96-well plate for 24 hours at 37 ° C. and 5% CO ₂ culture conditions. Incubated for RAW 264.7 cells were washed once with phosphate buffered saline (PBS) and replaced with fresh DMEM medium followed by LPS (1ug / ml) and LPS 1µg / ml, Comparative Example 1, Comparative Example 2 and Example 1 White rice extract and celecoxib were simultaneously treated at concentrations of 10 μg / ml, 50 μg / ml, 100 μg / ml, and 200 μg / ml and incubated for 20 hours. Dilute the supernatant was added to the PGE ₂ antibody plates (PGE ₂ antibody plate) and cultured for 18 hours or more at room temperature by treating the PGE ₂ -AchE teuraenseo (tracer). After incubation, the PGE ₂ antibody plate (PGE ₂ antibody plate) was washed 5 times with 0.05% Tween in PBS for 1 minute, and then incubated at room temperature for about 7 hours by treatment with Ellman's reagent. After absorbance was measured at 405 nm, the numerical value was substituted into the calibration curve prepared with PGE ₂ standard solution to convert the amount of PGE ₂ produced. PGE ₂ production inhibitory activity in the LPS-treated group was determined by non-linear regression analysis to determine IC ₅₀ (concentration that inhibits PGE ₂ production by 50%) of each test substance. The potency of the livers was compared.

As shown in Figure 6, when administration of the white rice extract of Comparative Example 1, Comparative Example 2 and Example 1 in RAW 264.7 mouse macrophage cells (prostaglandin) is one of the causes of inflammation As a result of comparing the concentration of E ₂ ), it was confirmed that Example 1 shows an excellent inhibitory effect corresponding to celecoxib.

Experimental Example 6. Effect of arthritis treatment using collagen-induced arthritis model

Measurement of arthritis intensification

6-1. How to measure

In order to examine the analgesic effect of arthritis for the above examples, formalin animal model experiments were conducted using the method described in the literature (Fazli-Tabaei S et al., Behav. Pharmacol., 16, pp613-619, 2005).

As a test animal, male ICR mice (Orient Bio, Japan) weighing 20 to 25 g were purified for several days, and 10 rats were used in each group. Drugs for each treatment group were administered orally, and after 1 hour, 10% formalin solution (Formalin, Sigma, USA) was subcutaneously administered to the left Fuji, immediately after injection for 5 minutes (1st phase) and 15 minutes to 20 minutes immediately after subcutaneous injection. Minutes (2nd phase) was recorded by measuring the time the mouse licking the soles, the inhibition rate (%) was expressed as a relative value based on the average time of the celecoxib treatment group 100.

division Concentration (mg / kg) % Inhibition 1st phase 2nd phase Comparative Example 1 400 90.4 75.9 Comparative Example 2 400 115.6 105.2 Example 1 400 126.4 124.1 Control Group (Celecoxib) 400 100 100

As shown in Table 1, the white rice extract of Example 1 showing anti-inflammatory activity in arthritis was superior to the analgesic inhibition rate compared to the white rice extract of Comparative Example 1 and the chloroform layer fraction of Comparative Example 2, selecock used as a positive control group It was confirmed that it is superior to the sheave.

In order to examine the analgesic effect of the above example, an arthritis animal model experiment induced by MIA (monosodium iodoacetate) was conducted using the method described in the literature (James D. Pomonis et al., Pain, 114, pp339). -346, 2005). As experimental animals, male SD rats (Oriental Bio, Japan) weighing 200-220 g were purified for several days, and then MIA (monosodium iodoacetate) (SIGMA, St. Louis, MO; cat # I2512) was dissolved in saline and left hind knee. Injection into the joint cavity caused arthritis and allowed a one week recovery period. One week later, an incapacitance tester (Linton, stoelting Co., Wood Dale, IL) was used to select individuals with arthritis, and to make up a group of 10 individuals per group. Samples are administered orally at the same time point every day from the 8th day after the induction of group separation, and measurement is started once a week for 3 weeks starting one week after the start of administration. The measurement was performed using an incapacitance tester (Incapacitance tester, Linton, stoelting Co., Wood Dale, IL), the measurement was performed using the following equation (1), the inhibition rate (%) is the average measured value of the celecoxib treatment group When expressed as 100, it is expressed as relative value.

division Concentration (mg / kg) Inhibition Rate (%)-3 weeks Comparative Example 1 400 76.1 Comparative Example 2 400 86.4 Example 1 400 100.7 Control Group (Celecoxib) 400 100

As shown in Table 2, the white rice extract of Example 1 showing anti-inflammatory activity in arthritis was superior to the analgesic inhibition rate compared to the white rice extract of Comparative Example 1 and the chloroform layer fraction of Comparative Example 2, selecock used as a positive control group It was confirmed that it is superior to the sheave.

Hot plate tests were performed using the method described in the literature as follows (Pharmacological report, 60 (2008) 409-414). As experimental animals, male ICR mice (Oriental Bio, Japan) of 15-20 g in weight were purified for several days, and 8-9 animals were used in each group. Each sample was orally administered, and after 1 hour and 2 hours, the mouse was placed in a plastic cylinder whose temperature was maintained at 55 ° C., and the time of licking or jumping of the hind paw was measured. The cut-off time of the mice was set to 15 seconds, and the inhibition rate (%) was expressed as a relative inhibition rate (%) calculated based on the inhibitory effect of celecoxib, a positive control group.

division Concentration (mg / kg) % Inhibition Comparative Example 1 400 85.2 Comparative Example 2 400 114.2 Example 1 100 132.1 200 133.5 400 143.2 Control Group (Celecoxib) 400 100

As a result, as shown in Table 3, the extract of Example 1 was tested for each concentration, the analgesic inhibition rate was excellent compared to Comparative Example 1, the extract of Example 1 is a single extract and Comparative Example 2, a chloroform layer fraction. In addition, it was confirmed to be superior to celecoxib used as a positive control.

To investigate the anti-inflammatory effect, acetic acid-induced litting assay animal model experiments were performed using the method described in the literature (HO J collier et al., Br. J. Pharmac. Chemother., 32, pp 295-310). , 1968).

As experimental animals, male ICR mice (Oriental Bio, Japan) of 20-23 g body weight were purified for several days, and 7-10 mice were used in each group. Samples of each treatment group were orally administered, and after 1 hour, 1% acetic acid solution (Acetic acid, Sigma, USA) was intraperitoneally administered, and 5 minutes after the injection, the number of times the mouse writhed was measured and recorded. Inhibition rate (%) was expressed as the relative inhibition rate (%) calculated based on the inhibitory effect of celecoxib, a positive control.

division Concentration (mg / kg) % Inhibition Comparative Example 1 400 31 Comparative Example 2 400 42.3 Example 1 100 65.4 200 67.1 400 79.3 Control Group (Celecoxib) 400 100

As a result, as shown in Table 4, the extract of Example 1 was tested for each concentration, the anti-inflammatory effect is excellent compared to Comparative Example 1, the extract of Example 1 is a single extract and Comparative Example 2, a fraction of the chloroform layer. Could confirm.

After male male Wistar rats (orients, males, 6 weeks old) were purified for several days, fasted from the day before the start of the experiment, weighed, divided into groups, orally administered samples at a concentration of 400 mg / kg per weight, and carrageenan dissolved in saline. Inflammation was induced by subcutaneous injection into the left foot of the hind leg. At regular intervals, plethysmometer was used to measure the swelling of the right and left paws without carrageenan injection. At this time, care was taken not to get foreign substances on the soles of the feet. In addition, celecoxib was orally administered at a concentration of 100 mg / kg per body weight as a positive control. The inhibition rate (%) was calculated based on the weight of the celecoxib administration group and is shown in Table 5 below.

division Concentration (mg / kg) % Inhibition Comparative Example 1 400 90 Comparative Example 2 400 92.3 Example 1 400 106.3 Control Group (Celecoxib) 400 100

As a result of measuring the swelling of the foot as described above, as shown in Table 5, the group orally administered the extract of Example 1 at 400mg / kg is superior to the group orally administered the anti-inflammatory drug celecoxib used previously I could confirm it.

Preparation Example 1 Preparation of Capsule

200 mg of the composite extract of Example 1

Lactose 50 mg

Starch 50 mg

Talc 2 mg

Magnesium stearate proper amount

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

Preparation Example 2 Preparation of Tablet

200 mg of white rice extract of Example 1

Lactose 100 mg

Starch 100 mg

Talc 2 mg

Magnesium stearate proper amount

Tablets are prepared by mixing and tableting the components according to conventional tablet production methods.

Preparation Example 3 Preparation of Liquid

1000 mg of white rice extract of Example 1

2000 mg of sugar

Isomerized sugar 2000 mg

Lemon flavor

Purified water is added to adjust the total amount to 1000 ml, and then the above ingredients are mixed according to a conventional liquid preparation method, and then filled into a brown bottle and sterilized to prepare a liquid preparation.

Preparation Example 4 Preparation of Health Functional Food

1000 mg of white rice extract of Example 1

<Vitamin mixture>

Vitamin A Acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Nicotinamide 1.8 mg

Folic acid 50 μg

Calcium Pantothenate 0.5 mg

<Inorganic mixture>

0.82 mg of zinc oxide

Magnesium carbonate 25 mg

Potassium citrate 90 mg

Calcium carbonate

The above ingredients may be mixed and granules prepared in accordance with a conventional health food preparation method, and may be used for preparing a health food composition according to a conventional method. The composition ratio of the above-mentioned vitamin and mineral mixture may be arbitrarily modified, and the above ingredients may be mixed according to a conventional health food manufacturing method, then granules are prepared and prepared in a health food composition according to a conventional method. Can be used.

Preparation Example 5 Preparation of Health Functional Drink

1000 mg of white rice extract of Example 1

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

900 mg total, including purified water

After mixing the ingredients according to a conventional healthy beverage production method, and stirred and heated at 80 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 ml container, sealed sterilization and refrigerated. Used to prepare healthy beverage compositions.

Preparation Example 6 Preparation of Capsule

200 mg of white rice extract of Example 1

50 mg of conventional arthritis medications (NSAIDs)

Lactose 50 mg

Starch 50 mg

Talc 2 mg

Magnesium stearate proper amount

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules. As the NSAIDs, known nonsteroidal anti-inflammatory drugs can be used, and the content thereof can be appropriately adjusted.

Preparation Example 7 Preparation of Ointment

1.0 wt% white rice extract of Example 1

20.0 wt% urea

15.0 wt% white petrolatum

Hard Flow Paraffin 6.0 wt%

Cetanol 3.0 wt%

Stearyl Alcohol 3.0 wt%

Glyceryl monostearate 5.0 wt%

Fragrance

Preservative

1.0 wt% buffer

Purified water remaining amount

The ointment is prepared by mixing the above components according to a conventional ointment preparation method.

Claims (5)

Adding 5 to 20 L of alcohol solvent per kg of dried root of Cinnanchum atratum, leaving it at 20 to 50 ° C. for 0.5 to 2 days, and then filtering and evaporating under reduced pressure to obtain an alcohol extract;
Adding a solvent in which water and hexane are mixed 1: 1 to the alcohol extract, and then extracting an organic material by adding chloroform to the separated water layer; And
Method for producing a pharmaceutical composition for preventing and treating arthritis, comprising the organic substance is fractionated by four steps of chromatography, each fraction is extracted by the step of extracting an effective fraction through an anti-inflammatory efficacy test as an active ingredient. The method according to claim 1,
The steroidal glucoside is a method for producing a pharmaceutical composition for preventing and treating arthritis, characterized in that the compound represented by the formula (1).
[Formula 1]

In which R is

or

to be. The method according to claim 1,
The alcohol solvent is a method for producing a pharmaceutical composition for preventing and treating arthritis, characterized in that ethanol or methanol. The method according to claim 1,
The chromatography is a method of producing a pharmaceutical composition for preventing and treating arthritis, characterized in that the one selected from the group consisting of normal phase vacuum flash chromatography, reverse phase open column chromatography and C18 reverse phase semi-prepared high performance liquid chromatography. delete

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