KR102048565B1 - Composition for Preventing or Treating Osteoarthritis comprising the complex extracts of allium hookeri and turmeric - Google Patents

Abstract

The present invention relates to a composition for preventing or treating osteoarthritis comprising a mixed extract of Allium hookeri and Curcuma longa. The mixed extract in which an Allium hookeri extract and a Curcuma longa extract is mixed in a weight ratio of 8:2 to 5:5, preferably 7:3, inhibits the expression of cytokines IFN-γ, TNF-α, IL-6, and IL-13 which are causes of arthritis, thereby having the activity for preventing and treating osteoarthritis. In addition, the mixed extract of Allium hookeri and Curcuma longa is a natural substance, so that it is harmless to the human body, and also has little toxicity and side effects, thereby being able to be safely applied for a long time as a pharmaceutical and food composition.

Description

Composition for Preventing or Treating Osteoarthritis comprising the complex extracts of allium hookeri and turmeric}

The present invention relates to a composition for preventing or treating osteoarthritis comprising a mixed extract of three and turmeric, and to prevent or treat osteoarthritis comprising a mixed extract of three and turmeric extracts mixed at a weight ratio of 8: 2 to 5: 5. It relates to a composition for.

Osteoarthritis is a chronic degenerative disease in which articular cartilage is gradually damaged by degeneration of extracellular matrix constituting articular cartilage, accompanied by inflammation and pain. Osteoarthritis occurs mainly in middle age or old age, and the prevalence of osteoarthritis in Korea accounts for about 40% in people over 50 years of age, and the prevalence of women is higher than that of men with increasing age. Osteoarthritis may be caused by degenerative changes, immune system abnormalities, infections, trauma and metabolic disorders. The factors related to the development of osteoarthritis include nitric oxide (NO), cytokines and proteolytic enzymes. Known.

Conventional treatment methods used to alleviate the pain of osteoarthritis include oral administration methods such as COX-1 or COX-2 inhibitors, narcotic analgesics, glucosamine (Glucosamin) and chondroitin (Chrondroitin), topical coatings, and steroids in the joint cavity. Topical administration methods such as injection of hyaluronic acid and the like have been implemented. In cases where the pain is relatively mild, drugs such as acetaminophen are used. However, despite the administration of acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs) are often used to treat arthritis in patients with persistent pain, severe pain, and inflammation. However, NSAIDs are limited in drug use because of the high risk of developing serious gastrointestinal complications in older people aged 65 or older, people who have had gastric ulcers in the past, those with gastrointestinal complications such as gastric bleeding, or who are on steroid or anticoagulant therapy. . In addition, glucosamine (Glucosamin) and chondroitin (Chondroitin) has been recommended for use in pain control of osteoarthritis patients in the European Rheumatology Society, but there is still much controversy about its stability. In the situation where all of the conventionally used chemical therapeutic drugs are showing limitations, various treatments for osteoarthritis based on various herbal ingredients have been developed in Korea.

Therefore, the present inventors have tried to develop a safe substance, especially plant-derived natural substance, which is safe for human body that can prevent or treat osteoarthritis related diseases, and as a result, the natural herbal composition comprising three and turmeric extracts as an active ingredient has anti-inflammatory action. The present invention was completed by elucidating that it is very effective for the prevention or treatment of osteoarthritis.

Therefore, the technical problem to be solved in the present invention is to provide a pharmaceutical composition for preventing and treating osteoarthritis comprising a naturally derived material.

In addition, the technical problem to be solved in the present invention is to provide a food composition for preventing and improving osteoarthritis comprising a natural derived material.

In order to solve the above technical problem, the present invention provides a pharmaceutical composition for osteoarthritis prevention and treatment comprising a mixed extract of three and turmeric as an active ingredient.

In addition, the present invention provides a food composition for preventing and improving osteoarthritis comprising a mixed extract of three and turmeric as an active ingredient.

Preferably, the mixed extract of three and turmeric is characterized in that the three and turmeric extract is mixed in a weight ratio of 8: 2 to 5: 5, more preferably 7: 3.

According to one embodiment of the invention, the composition comprising a mixed extract of three and turmeric as an active ingredient affects the progression of inflammation, differentiation of cells, destruction of joints, inflammatory cytokines that cause the development of arthritis It inhibits the expression of IFN- [gamma], TNF- [alpha], IL-6, and IL-13, thereby preventing or treating osteoarthritis.

Threeium ( Allium Hookeri ) used in the present invention is a plant that is known as a country of origin in Myanmar and is produced in India, China, etc. and widely used by local people, and in Korea, it is cultivated nationwide in 2012 after the trial cultivation in 2011. It's called ginseng because it has the shape of young ginseng and has three tastes (sweet, bitter and spicy). The three leaves and roots, also called root leeks, contain essential amino acids valine, isoleucine, methionine, threonine, lysine, phenylalanine, trytophan, Histidine and the like, and have a high content of vitamin A and vitamin C, nitrogen, phosphoric acid, iron, manganese, zinc and sulfur. In particular, sulfur content is more than six times more than garlic, and it is known that three Korean foods contain more than 700 mg of dietary sulfur per 100 g. Even if you do not hit the wild animals do not have the advantage of cultivation. In the type of sulfur component, the ginseng contains beta sulfur mainly, and the three compounds contain various types of sulfur components such as alpha, gamma, beta, delta, lambda, and mu sulfur.

Natural dietary sulfur contained in three vegetables helps to reduce cholesterol and break down blood clots, thereby helping to reduce diabetes, hyperlipidemia and lowering blood pressure, and have strong anti-cancer activity that reduces free radicals, harmful substances, remove inflammation, sterilization, diuresis and constipation. It has a variety of effects such as anti-aging, sperm and salivation, yang recovery, atopy, skin aging and exfoliation activity, strengthening bones and preventing hair loss.

Curcuma longa Rhizoma used in the present invention is a perennial plant belonging to the ginger tree, mainly cultivated in tropical and subtropical regions around India, and used for edible and medicinal stems and roots. Turmeric uses the root stem as a herbal medicine, and is a yellowish medicine with a hot and bitter taste. It is effective for pain relief and menstrual irregularities. In India, it is used as a medicine for bruises and sprains, or as a curry powder.

The term "extract" of the present invention refers to a formulation prepared by squeezing the herbal medicine with a suitable leach solution and evaporating the leach solution, but not limited thereto, but the extract obtained by the extraction treatment, the dilution or concentrate of the extract, the extract It may be a dried product obtained by drying, these adjustment products, or a refined product.

The three and turmeric extracts of the present invention can be extracted using conventional extraction solvents known in the art, and the extracted liquid can be used in liquid form or concentrated and / or dried. At this time, the extraction solvent is, for example, (a) water, (b) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) mixing the lower alcohol with water A solvent, (d) acetone, (e) ethyl acetate, (f) chloroform or (g) 1,3-butylene glycol can be obtained as an extraction solvent. Preferably, the extraction is performed using methanol, ethanol or butane. Depending on the extraction solvent, the extraction degree and loss degree of the active ingredient of the extract may vary, so select an appropriate extraction solvent. The extraction method is not particularly limited, and examples thereof include cold needle extraction, ultrasonic extraction, reflux cooling extraction, and the like.

In addition, the three and turmeric extract can be obtained through a conventional purification process in addition to the extraction by the extraction solvent. Obtained by various additional purification methods, such as separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity). Extracts can also be obtained through fractions.

According to an embodiment of the present invention, the three and turmeric extracts each contain about 0.5 to 20 times the weight of the three and turmeric samples, preferably 1 to 15 times the amount of water, C1 to C4 lower alcohols or water such as ethanol, methanol and the like. Using a mixed solvent of ethanol and an extraction method such as stirring extraction, hot water extraction, cold needle extraction, reflux cooling extraction or ultrasonic extraction for about 20 to 70 hours, preferably 40 to 50 hours at a high temperature, preferably The extract obtained after hot water extraction may be filtered, concentrated under reduced pressure or dried to obtain an extract.

The extract extracted with the solvent may be further fractionated with a solvent selected from the group consisting of butanol, hexane, methylene chloride, acetone, ethyl acetate, ethyl ether, chloroform, water and mixtures thereof.

The three and turmeric extract may be one obtained by completely removing the water by concentrating and freeze-dried the obtained extract, each extract that has completely removed the water is used in powder form or the powder is dissolved in distilled water or a common solvent Can be used.

The present invention is a composition having a osteoarthritis preventive effect containing a mixed extract of three and turmeric obtained by the production method as an active ingredient, the cytokine causes of osteoarthritis IFN-γ, TNF-α, IL-6 and IL-13 By inhibiting expression, a composition having prophylactic and therapeutic activity against osteoarthritis is provided.

In the present invention, the "active ingredient" alone means a component that can exhibit the desired activity or itself can exhibit activity with a carrier that is not active.

According to one embodiment of the invention, the mixed extract of the three and turmeric is preferably mixed in a weight ratio of 3: 7 to 7: 3.

In the composition of the present invention, the amount of the mixed extract of three and turmeric may be included in 0.001 to 20% by weight based on the total composition, more preferably the mixed extract of the three and turmeric may comprise 0.001 to 10% by weight based on the total composition. If it is less than 0.0001% by weight, the effect of inhibiting the expression of IFN-γ, TNF-α, IL-6 and IL-13 is too weak. If it exceeds 20% by weight, the effect of increasing the content is insufficient. There is a problem that the stability of the shape is not secured.

The composition containing the mixed extract of three and turmeric of the present invention exhibits an anti-inflammatory effect against osteoarthritis, and has little cytotoxicity as a natural substance.

According to one embodiment of the invention, it provides a pharmaceutical composition for the prevention and treatment of osteoarthritis comprising a mixed extract of three and turmeric.

As used herein, the term "prevention" refers to inhibiting the occurrence of a disease or condition in an individual who has not been diagnosed as having a disease or condition but is prone to such disease or condition.

As used herein, the term “treatment” means (a) suppression of the development of a disease or disorder in a subject (b) alleviation of a disease or disorder and (c) removal of the disease or disorder.

As used herein, the term "individual" refers to monkeys, cows, horses, pigs, sheep, dogs, cats, rats, mice, chimpanzees, and the like, including humans with diseases in which symptoms can be improved by administering the composition of the present invention. Means mammal.

A composition having osteoarthritis prophylactic and therapeutic activity according to one embodiment of the present invention comprises a pharmaceutically acceptable carrier in addition to a mixed extract of three and turmeric. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It is not. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans by various routes, such as oral or parenteral, for example, oral, rectal or intravenous, muscle, subcutaneous, intrauterine dural or intravascular. It can be administered by injection. It is preferably applied in the form of topical application during parenteral administration, more preferably by application.

Suitable dosages of the pharmaceutical compositions of the present invention may be prescribed in various ways depending on factors such as formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. The dosage of the pharmaceutical composition of the present invention may be administered once or several times a day in an oral dosage form of 0.001-100 mg / kg on an adult basis, and in an external preparation, 1.0 to 1 day in an adult basis. It is recommended that the amount be applied once to 5 times in an amount of 3.0 ml for 1 month or more. However, the dosage does not limit the scope of the present invention.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation may be used in any form suitable for pharmaceutical preparations, including powders, granules, tablets, capsules, suspensions, emulsions, syrups, oral formulations such as aerosols, external preparations such as ointments, creams, suppositories, and sterile injectable solutions. It may further comprise a dispersant or stabilizer.

On the other hand, according to one embodiment of the present invention, it provides a food composition for preventing and improving osteoarthritis comprising a mixed extract of three and turmeric.

In addition, the food composition according to one embodiment of the present invention, as an active ingredient, as well as a mixed extract of three and turmeric, as well as components commonly added in food production, such as proteins, carbohydrates, fats, nutrients, seasonings and Flavors may further be included.

Examples of such carbohydrates are monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As the flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

For example, when the food composition of the present invention is prepared with a drink, in addition to the mixed extract of three and turmeric of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, tofu extract, jujube extract, licorice extract and the like are added. It can be included as.

On the other hand, the mixed extract of the three and turmeric of the present invention is a natural substance, harmless to the human body, there is little toxicity and side effects, so it can be used safely even in the long-term use, in particular can be safely applied to the pharmaceutical and food compositions as described above have.

As such, the composition containing the mixed extract of three and turmeric according to the present invention as an active ingredient inhibits the expression of cytokines IFN-γ, TNF-α, IL-6 and IL-13 which are the causes of arthritis against osteoarthritis. Has prophylactic and therapeutic activity. In addition, the mixed extract of the three and turmeric of the present invention is a natural substance, harmless to the human body, and has little toxicity and side effects, and thus can be safely applied for a long time as a pharmaceutical and food composition.

Figure 1 shows the cell viability according to the extract of turmeric.
Figure 2 shows the cell viability according to the three extracts.
Figure 3 shows the cell viability according to the mixing ratio of the three and turmeric mixed extract.
Figure 4 shows the RNA expression changes of IFN-γ, TNF-α, IL-13 and IL-6 according to the mixing ratio of the three and turmeric mixed extract.
Figure 5 shows the protein expression changes of IFN-γ, TNF-α, IL-13 and IL-6 according to the mixing ratio of the three and turmeric mixed extract.
Figure 6 shows the established procedure of the arthritis animal model.
FIG. 7 shows the results of analysis of white blood cells (WBC), neutrophils (NE), eosinophils (EO), lymphocytes (LY) and monocytes (MO) in arthritis animal models.
Figure 8 shows the histopathological observation of arthritis animal model skin.
Figure 9 shows the expression changes of IFN-γ, TNF-α, IL-13 and IL-6 by three and turmeric mixed extract in arthritis animal model.
10 is histopathological observations of IFN-γ, TNF-α, IL-13 and IL-6 by three and turmeric mixed extract in arthritis animal model.

Hereinafter, examples and the like will be described in detail to help understand the present invention. However, embodiments according to the present invention can be modified in many different forms, the scope of the invention should not be construed as limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

Example 1 Preparation of Three Extracts

Three kg of three washes, dried after drying, pulverized and powdered, 1 L of purified water was added to 20 g of powder, and hot water extracted in a sterilizer, or extracted using 30% (v / v) EtOH and 70% (v / v) EtOH. The supernatant was concentrated using a concentrator to prepare three extracts.

Example 2 Preparation of Turmeric Extract

1 kg of turmeric was washed, dried, pulverized and pulverized, and then 1 L of purified water was added to 20 g of powder, and hot water extracted in a sterilizer, or extracted using 30% (v / v) EtOH and 70% (v / v) EtOH. The supernatant was concentrated using a concentrator to prepare a turmeric extract.

<Test Example 1> Identification of the bone joint improvement efficacy of three and turmeric in vitro )

(1) Verification of arthritis inhibitory effect using mouse macrophage

The change of cell viability and inflammatory cytokines according to solvents (alcohol, water) and the ratio of composite materials (three: turmeric = 0:10, 3: 7, 5: 5, 7: 3, 10: 0) The following experiment was conducted to show the significance.

① Efficacy verification through MTT assay (3- (4,5-dimethyl-2-thiazol) -2,5-tetrazolium bromide)

Put 100µl of RAW264.7 1 ㅧ 10 ⁴ cells / mL cells in a 96 well plate and incubate in 37 ℃ 5% CO ₂ incubator for 24 hours, and then process the concentration of each sample in each well. Incubated for 24 hours. 10 μl of 5 mg / ml MTT dissolved in PBS was treated in each well and incubated for 4 hours under the same conditions. Then, the culture solution was removed and 100 μl of DMSO was added to dissolve formazan formed at the bottom of the well, and then the absorbance was measured at 540 nm using a microplate reader.

Figure 1 shows the cell viability according to the extract of turmeric. As shown here, the cell viability of each extract was determined to be the most potent at 250 μg / mL of turmeric 30% EtOH extract.

Figure 2 shows the cell viability according to the three extracts. As shown here, the cell viability of each extract was measured and found to be effective at a concentration of 10 μg / mL of three 70% EtOH extracts.

Subsequently, 250 μg / mL of 30% EtOH extract and 10 μg / mL of three 70% EtOH extract, the concentrations of which are considered to be the most effective in cell viability of each of the three and turmeric, were selected and the mixing ratio of the three and turmeric extracts was selected. According to the cell viability was measured.

Figure 3 shows the cell viability according to the mixing ratio of the three and turmeric mixed extract. As shown here, the weight ratio of the three and turmeric was 7: 3.

② Efficacy verification through RT-PCR

The effects of three and turmeric extracts on the inhibition of IFN-γ, TNF-α, IL-6, IL-13 and β-actin genes and protein expression were tested.

First, RT-PCR was performed to investigate the effects of the mixed extracts of three and turmeric on IFN-γ, TNF-α, IL-6, IL-13 and β-actin gene expression. Total RNA was isolated from cartilage tissue using a total RNA extraction kit (iNtRON Biotechnology Inc., Korea). Each cDNA was synthesized from each total RNA. Using primers corresponding to the genes of IFN-γ, TNF-α, IL-6, IL-13, and β-actin, PCR cycle conditions were 2 min at 95 ° C, 5 seconds at 95 ° C → 30 ° C at 65 ° C. The seconds were repeated 40 times. Primers used for the RT-PCR are summarized in Table 1 below.

forward primer reverse primer IFN-γ 5'-AATGAACGCTACACACTGCA-3 ' 5'-TGAAGAAGGTAGTAATCAGG-3 ' TNF-α 5'-CCACATCTCCCTCCAGAAAA-3 ' 5'-AGGGTCTGGGCCATAGAACT-3 ' IL-6  5 '-TTGCCTTCTTGGGACTGATG-3' 5'-CAGAATTGCCATTGCACAACT-3 ' IL-13 5'-TCTGTGTAGCCCTGGATTCCC-3 ' 5'-CCGTGGCGAAACAGTTGCTT-3 ' β-actin 5'-GAAATCGTGCGTGACATC-3 ' 5'-GCTTGCTGATCCACATCT-3 '

       Figure 4 shows the RNA expression changes of IFN-γ, TNF-α, IL-13 and IL-6 according to the mixing ratio of the three and turmeric mixed extract. As shown here, the expression levels of IFN-γ, IL-13, and IL-6 were most decreased when the ratio of three and turmeric was 7: 3.

③ Efficacy verification through ELISA

ELSIA analysis analyzed cytokines of IFN-γ, TNF-α, IL-6, IL-13.

The capture Ab diluted in the coating buffer was added to the coat micro well, the cover was covered, overnight at 4 ° C., washed three times with the washing solution, and 200 μl of Assay Diluent was added to each well and incubated at room temperature for 1 hour. Subsequently, after washing, the standard and sample diluted with Assay Diluent were added to each well and incubated at room temperature for 2 hours.

Subsequently, washed five times, and added Working Detector (Detection Antibody + SAv-HRP reagent) to each well, incubated for 1 hour at room temperature, washed seven times, and incubated for 30 minutes under dark conditions at room temperature with TMB Substrate Solution. Stop solution was added to each well and measured at 450 nm.

Figure 5 shows the protein expression changes of IFN-γ, TNF-α, IL-13 and IL-6 according to the mixing ratio of the three and turmeric mixed extract. As shown here, the expression level of IFN-γ was most decreased when the mixing ratio of turmeric and three was 7: 3, and the expression level of IL-6 was the lowest when the mixing ratio of turmeric and three was 10: 0. , IL-13, TNF-α expression was most decreased when the weight ratio of three and turmeric was 7: 3.

<Test Example 2> Efficacy of improvement of bone joint improvement of samchae and turmeric in vivo )

(1) Establishment of arthritis animal model (air pouch arthritis animal model) and blood cell analysis through BALB / c

According to the path shown in Figure 6 after the animal obtained a period of 7 days after the period of group separation was carried out. 2 mL of air was injected into the intra-scapular three times at two-day intervals. Three days after the injection, a positive control group, methylsulfonylmethane (MSM), and three or turmeric mixtures (7: 3 weight ratio) by concentration were administered orally. Figure 6 shows the established procedure of the arthritis animal model.

Carageenan was administered 2 hours after oral administration and autopsy was performed 24 hours later.

At necropsy, skin and exudate were collected and analyzed using a blood analyzer (Drew Scientific, Inc., Oxford, USA).

7 is a result of analysis of white blood cells (WBC), neutrophils (NE), eosinophils (EOs), lymphocytes (LY) and monocytes (MO) in the arthritis animal model. As shown here, hemocytosis of the exudate decreased the concentration-dependently in the administration groups of the three and turmeric mixtures compared to the positive control group, MSM, and showed similar efficacy as the positive control group. In addition, it was confirmed that the reduction of more than 10% from 500 mg / kg compared to the positive control.

(2) Efficacy verification through histopathology

The skin obtained above was subjected to the removal and hydration of paraffin (xylene 5 min → xylene 5 min → xylene 5 min → 80% EtOH 1 min → 90% EtOH 1 min → 100% EtOH 1 min).

After dehydration, water was rinsed in tap water for 5 minutes, wiped well, and soaked for 1.5 minutes in hematoxylin solution. After staining, the tissues were washed in running water until they looked blue.

Clarification process and dehydration process (95% EtOH 5 min → Eosin 5 min → 95% EtOH 1 min → 95% EtOH 1 min → 100% EtOH 1 min → 80% EtOH 1 min → 90% EtOH 1 min → 100% EtOH 1 min → xylene 6 min) and then tissue loading by mounting process.

Figure 8 shows the histopathological observation of arthritis animal model skin. As a result of H & E staining of skin of arthritis animal model, air pouch membrane was thinned in carrageenan group compared to CON and similar to CON in MSM group. In the three and turmeric mixtures, the air pouch membrane was widened depending on the concentration. As a result, the efficacy of the positive control MSM and the three / turmeric mixture was similar.

(3) Efficacy verification through change of inflammation-related indicators

① Efficacy verification through exudate (ELISA)

ELSIA analysis analyzed cytokines such as IFN-γ, TNF-α, IL-6, IL-13. The capture Ab diluted in the coating buffer was added to the coat micro well, and the cover was covered and overnight at 4 ° C. After washing three times with the washing solution, 200 ㎕ of Assay Diluent was added to each well and incubated at room temperature for 1 hour.

After washing, put the standard, sample diluted with Assay Diluent in each well and incubated for 2 hours at room temperature. After washing 5 times, working detector (Detection Antibody + SAv-HRP reagent) was added to each well and incubated for 1 hour at room temperature, and then washed 7 times, and TMB substrate solution was added to each well and incubated for 30 minutes under dark conditions at room temperature. A stop solution was added to each well and measured at 450 nm.

Figure 9 shows the expression changes of IFN-γ, TNF-α, IL-13 and IL-6 by three and turmeric mixed extract in arthritis animal model. As shown here, ELISA analysis of inflammatory cytokines using exudate resulted in a concentration-dependent decrease in the cytokine expression levels in the three and turmeric mixtures compared to the positive control group, MSM, and IFN compared to the positive control group. From 250 mg / kg of -γ it was confirmed that a significant decrease over 60%.

② Validation through organization (IHC)

Paraffin removal and hydration (xylene 5 min → xylene 5 min → xylene 5 min → 80% EtOH 1 min → 90% EtOH 1 min → 100% EtOH 1 min) were performed. After running for 3 minutes after washing with distilled water and maintained for 40 minutes in a bath at 90 ℃-100 ℃ for antigen retrieval process. After standing at room temperature for 5 minutes, holding for 10 minutes in cold water and washing was repeated twice for 5 minutes with TBS-T.

After immunoblocking (Immunoblocking) it was covered with parafilm and reacted at room temperature for 1 hour and washed twice with TBS-T for 5 minutes. After diluting the primary antibody with blocking serum, it was placed on the tissue, covered with parafilm, and reacted at room temperature for 30 minutes.

After the reaction, washed twice with TBS-T for 5 minutes, and the secondary antibody was placed on the tissue and reacted for 10 min. After the reaction was washed twice with TBS-T for 5 minutes, and after performing the chromogen reaction to confirm the reaction degree in the appropriate state in PBS to stop the reaction and then washed twice with TBS-T for 5 minutes.

After washing, hematoxylin was used for staining the counter, and the reaction was stopped by distilled water. The reaction was washed twice with TBS-T for 5 minutes.

Clarification process and dehydration process (95% EtOH 5 min → eosin 5 min → 95% EtOH 1 min → 95% EtOH 1 min → 100% EtOH 1 min → 80% EtOH 1 min → 90% EtOH 1 min → 100% EtOH 1 min → xylene 6 min) and then tissue loading by mounting process.

10 is histopathological observations of IFN-γ, TNF-α, IL-13 and IL-6 by three and turmeric mixed extract in arthritis animal model. As shown here, the immunostaining of inflammatory cytokines on the skin was progressed, and as a result of analysis, the expression level of each cytokine was increased in the carrageenan group compared to CON, and in the three and turmeric mixture concentration groups compared to the positive control group MSM. The expression level of each cytokine decreased in a concentration-dependent manner, and showed similar effects as the positive control group.

Claims (6)

Osteoarthritis prevention or treatment pharmaceutical composition comprising a mixture of three and turmeric as an active ingredient. The method of claim 1,
The mixed extract of the three and turmeric is three pharmaceutical extracts and turmeric extract is a pharmaceutical composition for preventing or treating osteoarthritis, characterized in that the mixture in a weight ratio of 8: 2 to 5: 5. The method of claim 2,
The three extract and turmeric extract is a pharmaceutical composition for the prevention or treatment of osteoarthritis, characterized in that the mixture in a weight ratio of 7: 3. Osteoarthritis prevention or improvement food composition comprising a mixture of three and turmeric as an active ingredient. The method of claim 4, wherein
The mixed extract of the three and turmeric is a food composition for preventing or improving osteoarthritis, characterized in that the three extract and turmeric extract is mixed in a weight ratio of 8: 2 to 5: 5. The method of claim 5,
Osteoarthritis prevention or improvement food composition, characterized in that the three extract and turmeric extract is mixed in a weight ratio of 7: 3.

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