KR102142342B1 - Pharmaceutical composition for arthritis - Google Patents

Abstract

Provided is a pharmaceutical composition for preventing or treating arthritis, which comprises a mixture of egg yolk and bamboo salt as an active ingredient.

Description

Pharmaceutical composition for prevention or treatment of arthritis {PHARMACEUTICAL COMPOSITION FOR ARTHRITIS}

The present invention relates to a pharmaceutical composition for the prevention or treatment of arthritis.

Arthritis is a chronic degenerative disease, in which joint cartilage damage and cartilage cell death occur due to inflammation in the joint. It is the most common form of joint disease, showing a prevalence rate of 5% of the world's population, and a prevalence rate of 10.7% in 2008 in Korea. In particular, with the rapid increase in the elderly population and the prevalence of obesity, arthritis patients also tend to increase, and the prevalence of up to 34.5% is over 80 years old.

The exact cause of the development of arthritis has not been identified, but factors such as physical stress caused by obesity such as sulfuric acid and cytokines are known to be closely involved in the development. Currently, clinically used degenerative arthritis treatment methods include analgesics, steroids, nonsteroidal anti-inflammatory drugs, and cartilage protection agents such as clocosamine and hyaluronic acid, or surgical treatment. In the case of drug treatment, the pain or inflammatory response itself is non-specifically relieved, and the cartilage protector acts to protect the joints by affecting chondrocytes or alleviating shock. Among them, steroid-based drugs have side effects that can cause diseases such as osteoporosis, hypertension, and diabetes due to loss of calcium when taken for a long time. In this way, the drug treatment of arthritis is mostly used only for the purpose of reducing pain, and surgical methods such as permanent artificial joint replacement surgery are mainly used, and there are no drugs showing fundamental therapeutic effects to date.

The market for degenerative arthritis treatment is growing every year. In 2014, the market for analgesic anti-inflammatory drugs and hyaluronic acid therapeutics, which are therapeutic drugs, amounted to 21 billion dollars, accounting for 2.8% of the global pharmaceutical market of 490 billion dollars. Artificial joints accounted for $14.1 billion, accounting for 3.76% of the global medical device market of $37.52 billion.

If you look at the health insurance data from 2011 to 2014 in Korea, degenerative arthritis treatment expenses averaged 2% per year from 1 trillion 11.3 billion won in 2011 to 1 trillion 3162 billion in 2014, and the number of patients averaged from 4.47 million in 2011 to 4.41 million in 2014. It is predicted that the market will increase steadily as it shows a trend of increasing %.

Chondrocytes are the only cells present in articular cartilage and are deeply involved in the development of arthritis. The death of chondrocytes breaks the balance of cartilage synthesis and absorption processes, resulting in loss of cartilage and eventually structural and functional abnormalities of cartilage. It is known that interleukin 1 beta (IL-1β), Tumor necrosis factor alpha (TNF-α) and NO are deeply involved in the death of chondrocytes. It is known that IL-1β inhibits extracellular matrix synthesis and chondrocyte differentiation, and TNF-α inhibits mesenchymal cells from differentiating into chondrocytes, resulting in cartilage loss. In addition, according to previous research results, it was confirmed that high concentrations of Nitric oxide (NO) and Prostaglandine E2 (PGE2) exist in the joint lubricating tissue of arthritis patients, and these NO and PGE2 stimulate IL-1β by stimulating chondrocytes. It is known to appear. NO plays an important role in blood vessel tension and neurotransmitter immune function and is known to produce excessive amounts during the process related to the development of osteoarthritis. In addition, it is known to cause inflammatory reactions such as tissue damage, genetic variation, and edema. . In addition, PGE2 is recognized as the most important substance in the pathogenesis of inflammation, and is known to be rapidly produced in macrophages when inflammation occurs. NO plays a role in the early stages of leukocyte migration to the inflammatory site, and PGE2 is a substance that mainly acts on the posterior part where fever and pain appear.

Background art of the present invention is described in Korean Patent Publication No. 2016-0019006.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating arthritis.

The pharmaceutical composition of the present invention includes a mixture of egg yolk and bamboo salt, and may have a purpose of preventing or treating arthritis.

The present invention provides a pharmaceutical composition for preventing or treating arthritis.

1 is a result of measuring the concentration of IL-1β and TNF-α in the blood in the experimental example.
2 is a result of measuring the concentration of NO and PGE2 in the blood in the experimental example.
Figure 3 is a photograph of the efficacy of arthritis by histological examination in the experimental example.
4 is a test result of DPPH radical scavenging activity in the experimental example.
Figure 5 shows the results of the hydroxy radical scavenging activity in the experimental examples.
6 is a result of evaluating the severity of arthritis in the experimental example.
7 is a photograph taken in the experimental example.
8 is a concentration result of the anti-collagen antibody in the experimental example.

Embodiments will be described in detail with reference to the accompanying drawings so that those skilled in the art to which the present invention pertains can easily practice. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. In the drawings, parts not related to the description are omitted in order to clearly describe the present invention, and the same reference numerals are assigned to the same or similar elements throughout the specification.

The pharmaceutical composition of the present invention may include a mixture of egg yolk and bamboo salt as an active ingredient.

It is preferred that the egg yolk is prepared by baking and heating the high-cigarette by heating the broth (natural mineral containing CuSO ₄ ·5H ₂ O) as a main component, and powdering the high-cigarette, followed by mixing and mixing egg whites of eggs. In this way, the egg yolk half is neutralized by the egg whiteness of the egg, and the toxicity is reduced or eliminated and the drug is enhanced. The egg board prepared in this way may be in powder form. The egg yolk board may be included in the pharmaceutical composition in a powder form, but may also be included by dissolving the powder in a predetermined solvent.

The method of manufacturing the egg yolk is specifically described. Egg yolk half (a) heating the natural mineral crude drug Damban (CuSO ₄ ·5H ₂ O) until the total amount turns gray; (b) cooling the heated damban and pulverizing it; And (c) mixing the light powder of step (b) with egg whites.

First, step (a) is a step of heating damban (a natural mineral containing CuSO ₄ ·5H ₂ O). As a method of heating the tobacco, a conventional heating method known in the art can be used, and preferably, it is placed in a cauldron and can be heated by a method of baking in a gas fire, wood fire or charcoal fire. When heating, it is good to mix the top and bottom, evenly watching the color change every 3~5 hours, so that it receives the heat evenly. The heating time can be adjusted according to the amount used once, but it is suitable to heat for about 10 to 24 hours, and it is preferable to heat until the color of the entire amount of light brown is changed to gray and becomes dehydrated.

Step (b) is a step of cooling the heated tobacco in step (a), followed by powdering to prepare a high tobacco. The heated damban must be cooled until the heat is completely exhausted. At this time, the moisture content of the damban is preferably about 0% to 5% by weight. After the heated dish is completely cooled, it is finely ground to powder, and it is preferable to store the powdered dish in a plastic bag or airtight container in a dry place so that moisture is not absorbed.

Step (c) is a step of reducing or removing the toxicity of the high-cigarette by increasing the high-cigarette powder of the step (b) to the egg white egg and reacting the high-cigarette with the egg white egg, thereby enhancing the weakness. Eggs use egg eggs by separating egg yolk and egg white, and it is preferable to use native eggs, and more preferably eggs are egg bones. The egg whites are mixed with the heated high-fat powder prepared in the step (b), wherein the mixing ratio is 7 to 20 egg whites (140 g to 700 g, preferably 200 g to 700 g) per 600 g of light powder. It is preferred to mix. When mixing, it is good to control the amount of egg whites while looking at the condition and mix them using a tool that has little reactivity, such as a wooden spatula. In addition, it is preferable to mix using a container in which a chemical reaction does not occur, for example, a ceramic bowl, porcelain, or mackban stone container. Please note that high heat is generated by the heat of reaction during the mixing process. If the amount of egg whites during mixing is too small, it is difficult to sufficiently neutralize the toxicity of the heated pale powder. On the other hand, when mixing, the amount of egg whites is difficult to obtain because the reaction heat is weak or does not occur when mixing. Adjust it carefully. After sufficiently mixing the above mixture, it is preferable to cool the heat until the reaction heat generated during the reaction completely disappears.

The method of preparing the egg yolk may further include, after step (c), (d) cooling the mixture of step (c) and then pulverizing. Step (d) is a step in which the mixture of step (c) is cooled until heat is completely discharged, and then the mixture of egg yolks is ground to finely powder. By powdering the egg yolk, it can be effectively used in pharmaceutical or food preparations, and the reactive area of the egg yolk is increased to maximize disease therapeutic activity.

Bamboo salt can be used commercially or can be prepared directly. Bamboo salt is made of bamboo salt by the inventor of the salt inventor, Kim Il-hoon, in the booklet'Space and New Drugs (1980)' and New Drug (1986). When the bamboo tree burns out, the remaining salt column is crushed, put into the new bamboo tree as the first one, and then it is capped with ocher to repeat the burning of the pine tree from the iron barrel eight times. It is preferable to use bamboo salt that is faithful to the authentic 9-time molten bamboo salt manufacturing method of the melted phosphoric acid teacher, but is not limited thereto. Bamboo salt may be included in the pharmaceutical composition in a powder form, but may also be incorporated by dissolving the powder form in a predetermined solvent. Bamboo salt may include purple-colored autism.

The pharmaceutical composition may include a mixture of egg yolk and bamboo salt. The above-mentioned mixture may be prepared by mixing separately prepared egg yolk and bamboo salt. The weight ratio of the egg mass: bamboo salt in the mixture may be 1:1 to 1:100, preferably 1:10 to 1:60, and more preferably 1:10 to 1:30. In the above range, the effect of treating arthritis is high and side effects may be small.

The pharmaceutical composition is useful for the prevention or treatment of arthritis by including a mixture of egg yolk and bamboo salt. As used herein, "prevention" means any action that suppresses the formation of a disease or delays the onset of the administration of a composition, and "treatment" means any action that improves or beneficially alters the symptoms of the disease through administration of the composition. It means. In the present application, "arthritis" may mean degenerative arthritis or rheumatoid arthritis.

The pharmaceutical composition may be used as a pharmaceutical composition by further including a pharmaceutically acceptable carrier, and may be formulated with a carrier. In addition, the composition of the present invention can be used as a single agent or in combination with other active ingredients that can enhance drug efficacy. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound. As a pharmaceutical carrier acceptable in a composition formulated as a liquid solution, as a biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and these components One or more of them may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets.

The pharmaceutical composition can be applied to any formulation containing a mixture of egg yolk and bamboo salt as an active ingredient, and can be prepared in oral or parenteral formulations. The pharmaceutical formulation of the present invention is administered by oral, rectal, nasal, topical, subcutaneous, vaginal or parenteral (including intramuscular, subcutaneous and intravenous). And suitable forms for administration by inhalation or insufflation. Formulations for oral administration comprising the composition of the present invention may be formulated as, for example, tablets, troches, lozenges, water-soluble or oily suspensions, preparation powders or granules, emulsions, hard or soft capsules, syrups or elixirs. . For formulation into tablets and capsules, formulations such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, magnesium stearate , May include a lubricant such as calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax, and in the case of capsule formulations, may further contain a liquid carrier such as fatty oil in addition to the above-mentioned substances. Formulations for parenteral administration comprising the composition of the present invention may be formulated for injection, such as subcutaneous injection, intravenous injection, or intramuscular injection, suppository injection method, or aerosol preparation such as inhalation through the respiratory system, for spraying Can. In order to formulate an injectable formulation, the composition of the present invention may be prepared as a solution or suspension by mixing in water with a stabilizer or buffer, and formulated for unit administration of ampoules or vials. To inject into a suppository, it can be formulated into a composition for rectal administration such as a suppository or a body enema containing a conventional suppository base such as cocoa butter or other glycerides. When formulated for spraying, such as aerosols, propellants and the like may be combined with additives to disperse the concentrated dispersion or wet powder.

The pharmaceutical composition can be used for the prevention or treatment of arthritis. It will be apparent to those skilled in the art that a suitable total daily dosage can be determined by the treating physician within the proper medical judgment. The specific therapeutically effective amount for a specific patient is the specific composition, patient age, weight, general health status, gender and diet, time of administration, administration, including the type and extent of the reaction to be achieved and, in some cases, whether other agents are used. It is desirable to apply differently depending on various factors including the route and secretion rate of the composition, the duration of treatment, the drug used with or concurrently with the specific composition, and similar factors well known in the pharmaceutical field. Therefore, the effective daily dosage of the pharmaceutical composition suitable for the purpose of the present invention is preferably determined in consideration of the above, most preferably 0.007g/kg to 0.007g/kg when taken on the basis of a mixture of egg yolk and bamboo salt It is about 0.3g/kg and there is no limit to the amount for use as an external preparation such as enema or application.

The pharmaceutical composition is applicable to any animal in which arthritis may occur, and the animal includes humans and primates, as well as livestock such as cattle, pigs, sheep, horses, dogs, and cats. Particularly, when orally administering the composition of the present invention, a method of swallowing with decoction by mixing water, ginger, or licorice, a method of swallowing with a root extract, or a method of swallowing with saliva is possible. In addition, in addition to just eating, there is also a method of taking the capsule in several ways as described above, and swallowing the capsule with saliva is more effective than swallowing with water or drink. You can also mix it with food or drinks in small amounts. For example, there is a method of adding the above mixture to porridge or roasted garlic, and there is also a method of mixing the bamboo salt with soy sauce powder.

A mixture of egg yolk and bamboo salt may be used as an active ingredient in health functional food for preventing or improving arthritis. Health functional food may be formulated by further including a food additive that is food-acceptable, and may be used as a health functional food for preventing and treating arthritis. The health functional food of the present invention includes tablets, capsules, pills, liquids, and the like. Foods to which the composition of the present invention can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, and health functional foods. "Health functional food" as defined herein refers to food manufactured and processed using raw materials or ingredients having useful functionality for the human body according to the Act No. 6727 on the Health Functional Food, and "functional" means It means to ingest for the purpose of obtaining useful effects for health purposes such as adjusting nutrients for structure and function or physiological action. The composition of the present invention may be added to a food or beverage for the purpose of preventing or improving arthritis, wherein the amount of the extract in the food or beverage can be added at 0.01 to 10% by weight of the total food weight, healthy beverage composition It can be added at a rate of 0.01 to 5 g, preferably 0.5 to 1 g, based on 100 ml of silver.

Hereinafter, the configuration and operation of the present invention through a preferred embodiment of the present invention will be described in more detail. However, this is provided as a preferred example of the present invention and cannot be interpreted as limiting the present invention by any means.

Board Produce

Godamban (baked damban) is produced by heating raw tobacco. If you bake it by heating it for 24 hours, it becomes gray while heating it. This condition of dehydration is called godamban. After the godamban was completely cooled, the godamban was prepared by finely grinding and pulverizing.

Nandamban is prepared by mixing egg white powder with egg white powder and reacting it. Gotban powder powder 600g, 13~25 eggs from native eggs are separated and mixed with 200g~700g egg white. Gothamban reacts with moisture in the egg white to turn green when high heat (heat of reaction) occurs and the powder becomes tangled. , This condition is called the gang.

Apical salt Produce

The sea salt, minus the brine, was chopped into a bamboo node and the bamboo was covered with water-blown ocher. The bamboo salt was chopped into bamboo salt furnace and baked at 800℃ with pine wood fire. The bamboo that was baked in the bamboo salt furnace was turned into ash, and the bamboo and loess were removed, and the salt that had become a salt pillar was taken and crushed.

The salt obtained by crushing was re-filled in bamboo, and the above-described process of blocking with ocher and baking at 800°C was repeated 8 times. The resulting crushed salt was heated and melted, and the liquefied rosin was heated to 1300°C while blowing air with fuel to melt the salt mass and flowed into the liquid to remove ashes and silicates to obtain sucrose.

Preparation of egg yolk and apricot salt mixture

Using the prepared egg yolk half and autitis salt, 1 g of egg yolk half and 20 g of autism salt (weight ratio of egg yolk half: porridge salt were 1:20) were obtained, thereby obtaining a mixture of egg yolk half and aquatic salt.

<Experimental Example>

Evaluation of pharmaceutical efficacy in degenerative arthritis animal model of composition

end. Construction of an animal model with degenerative arthritis

Male SD rats weighing 210 g to 240 g were purchased from Daehan Biolink and managed in an animal breeding facility where constant environmental conditions were maintained, and healthy animals were selected and used in the production of disease models. Degenerative arthritis was caused by dissolving 4 mg of monosodium iodoacetate (MIA), which inhibits the activity of glyceraldehyde-3-phosphate hydrolase in vivo, in 50 μl of physiological saline and administering it to the animal's knee joint cavity. MIA suppresses the process, causing destruction of articular cartilage and death of chondrocytes. These animal models are very similar in histology to human arthritis, and have been used in previous studies to verify the efficacy of candidate drugs for the treatment of arthritis.

I. Administration of the composition

According to the results of previous studies and preliminary experiments, it was confirmed that after 3 weeks of administration of MIA, grades 3 to 4 of degenerative arthritis developed.

Therefore, the composition was administered 3 weeks before administration of MIA to the animal model, and the composition was orally administered to animals for a total of 6 weeks until 3 weeks after administration of MIA. The experimental group consists of a total of 5 groups as shown in Table 1 below:

①Normal group: A group that did not develop degenerative arthritis because MIA was not administered

② Disease-causing group: A group in whom degenerative arthritis developed due to administration of MIA, and no degenerative arthritis treatment was administered.

③ Nandamban administration group: MIA was administered to develop degenerative arthritis and the group to which the Nandamban was administered. The egg yolk was administered orally.

④ bamboo salt administration group: MIA was administered to develop degenerative arthritis, and the group was treated with autitis. Autism was administered orally.

⑤Complex administration group: A group in which degenerative arthritis developed due to administration of MIA and a mixture of egg yolk plaque and autitis. The mixture of egg yolk and bamboo salt was administered orally.

Test group Dosage concentration
(mg/kg) Dosing period
(week) Number of animals Before induction After induction Normal - - - 4 Disease incidence - - - 6 Egg group 10 3 3 6 Bamboo salt administration group 200 3 3 6 Combined administration group 200 3 3 6

The composition was administered once a day at 24 hour intervals.

All. Changes in body weight and blood biochemical levels of the animal model upon administration of the composition

I. According to the item, the body weight of the animals was measured once a week while the composition was administered orally for a total of 6 weeks. After 10 hours after the final administration of the composition, AST, ALT, and BUN and CRE were measured as items related to liver function from serum obtained from the animal model, and the results are shown in Table 2 below.

Test group AST ALT BUN CRE IU/L IU/L mg/dL mg/dL Normal 112.4±13.5 53.4±7.4 17.7±2.1 0.4±0.1 Disease-causing group 123.6±23.4 63.4±13.6 18.1±2.5 0.4±0.1 Egg group 129.4±30.5 65.3±16.5 17.8±2.1 0.4±0.0 Bamboo salt administration group 115.3±20.4 63.9±10.3 18.2±2.0 0.4±0.1 Combined administration group 110.6±18.4 57.3±11.5 17.9±2.0 0.4±0.0

As in the above results, no change was found to be due to the composition in liver and kidney related blood tests. This can be an indirect indicator that the composition does not cause problems with liver and kidney function in experimental animals, and can be indirectly judged to be non-toxic.

la. Blood IL- in animal models 1β and TNF -α concentration measurement

After the administration of the composition for a total of 6 weeks was completed, an experimental animal was anesthetized with carbon dioxide and opened and blood was collected from the abdominal aorta. The obtained blood was left at room temperature for 30 minutes, and then centrifuged at 3000 rpm for 10 minutes to obtain serum. In the obtained serum, the concentrations of IL-1β and TNF-α in cytokine were measured using an enzyme-linked immunosorbent assay (Enzyme-Linked ImmunoSorbent Assay, ELISA). The results are shown in FIG. 1. As shown in FIG. 1, as a result of confirming the cytokines IL-1β and TNF-α, which play a major role in the development of arthritis, meaningful results could be found in the group administered with the egg bladder and bamboo salt complex. These results can be said to mean that the composition has the effect of alleviating arthritis through cytokine control.

hemp. Measurement of blood NO and PGE2 levels

The concentrations of NO (nitric oxide) and PGE2 (prostaglandin E2) were measured in serum of experimental animals in which a total of 6 compositions were administered. NO plays an important role in blood vessel tension and neurotransmitter immune function and is known to produce excessive amounts during the process related to the development of osteoarthritis. In addition, it is known to cause inflammatory reactions such as tissue damage, genetic variation, and edema. . In addition, PGE2 is recognized as the most important substance in the pathogenesis of inflammation, and is known to be rapidly produced in macrophages when inflammation occurs. NO plays a role in the early stages of leukocyte migration to the inflamed area, and PGE2 mainly acts in the latter part of the appearance of fever and pain. The results are shown in FIG. 2. As shown in FIG. 2, it was possible to find meaningful results in the group where the egg block and bamboo salt were combined. These results can be said to mean that the composition has the effect of alleviating arthritis through the control of inflammatory mediators such as NO and PGE2, which play a major role in the development of arthritis.

bar. Histological examination measures the efficacy of the composition against arthritis

Knee joints were collected after the 6-week period of composition administration, and fixed in 10% neutral buffered formalin. After 48 hours of demineralization using 5% formic acid, a paraffin block was produced through a general tissue separation process, and a 4 micrometer-thick tissue slide was prepared using a thinning machine. Using the prepared slide, H&E staining was performed to check for changes in chondrocytes in the knee joint and inflammatory cell infiltration, and Safranine O staining was performed to observe the damage state of the knee cartilage. The results are shown in FIG. 3. As shown in FIG. 3, arthritis was induced in the disease-producing group compared to the normal group, and it was confirmed that the joint tissue was significantly deformed and the damage of chondrocytes occurred significantly. Could.

four. Evaluation of the antioxidant efficacy of the composition (1)

It is known that free radicals in the body attack cells and become the main cause of various diseases and aging, and also affect the development or progression of degenerative joint diseases. DPPH has a chemically stable free radical, and when it encounters a substance with antioxidant activity, the electron is given and the radical is extinguished and the color change from purple to yellow was used to confirm the antioxidant efficacy of the composition.

180 μl of 20 μM DPPH solution dissolved in ethanol in a 96-well plate and 20 μl each at a final concentration of 1, 3, 10, 30, 100, 300, 1000 μg/ml, and 37 μg of light-shielded state After incubation at 20 minutes, the absorbance at 517 nm was measured using a FL 600 spectrofluorometer (Bio-Tek, USA). The results are shown in FIG. 4.

As shown in FIG. 4, when tested to a concentration of 1000 micrograms per milliliter maximum, when using only light weight, although the concentration increased slightly, it showed only an effect of 22.8% even at the maximum concentration. When only bamboo salt was used, it showed 84.9% radical scavenging ability at a concentration of 300 micrograms per 1 liter. When used in combination, it showed superior efficacy than before, showing a maximum scavenging activity of 96.4% at a concentration of 300 micrograms per milliliter.

Ah. Evaluation of the composition's antioxidant efficacy (2)

The hydroxyl radical is the most dangerous free radical, composed of the same ratio of hydrogen and oxygen, and when it comes into contact with other molecules at a rate of 100 millionths of a second, it attacks its own lone electron by a momentary attack. The structure in which one oxygen atom is combined with one oxygen atom is denoted by -OH, and the change in the amount by the composition is measured using an absorbance meter to confirm the antioxidant effect of the composition.

The scavenging capacity of non-site specific hydroxyl radicals (OH·) of each sample solution was measured by modifying the method of Halliwell et al. (1987). Specifically, 28 mM deoxyribose, so that the final volume was 1 mL for samples of various concentrations, 14 mM hydrogen peroxide, 200 μM FeCl 3, 1 mM EDTA, 10 mM ascorbic acid and potassium phosphate buffer (pH 7.4) were added. After mixing the mixture and reacting at 37°C for 1 hour, 1 mL of 10% (w/w) trichloroacetic acid (TCA) solution was added to stop the reaction, and 1% (w/w) of TBA (2-thiobarbituric acid) was added thereto. ) The solution was added, heated at 100° C. for 15 minutes, rapidly cooled, and absorbance was measured at 532 nm. The results are shown in FIG. 5.

As shown in FIG. 5, when tested to a concentration of up to 1000 micrograms per milliliter, only a small amount increased as the concentration increased, but only a 12.2% hydroxyl radical reduction effect was observed even at the maximum concentration. When only bamboo salt was used, a hydroxyl radical reduction effect of 33.6% was observed at a concentration of 300 micrograms per milliliter. When used in combination, it showed superior efficacy than before, showing a hydroxyl radical reduction effect at a concentration of 300 micrograms per milliliter.

Compositional Rheumatism Evaluation of pharmaceutical efficacy in an animal model of arthritis

The effect of the mixture of egg yolk and autism (1:20 weight ratio) on the animal model of rheumatoid arthritis induced by collagen was examined.

1) Rheumatoid arthritis mouse model production

In order to produce an animal model of collagen-induced arthritis, a collagen solution in which 2 mg/ml of type 2 collagen was dissolved and an equal amount (w/v) of Freund's complete adjuvant (Chondrex, SA, USA) were mixed. After sensitization, 0.1 ml was administered subcutaneously using a 1 ml syringe. After another two weeks, Freund's incomplete adjuvant was mixed in the same amount with the collagen solution prepared by the same method, and the mice's tail was injected subcutaneously into the buttocks leading to the body, thereby causing rheumatoid arthritis. Each experimental group used 8 mice per group, a normal control that did not cause arthritis (Normal control), a control that caused arthritis (CIA-control), and a group that received arthritis and then administered Dexamethasone (Dexa-control) ), and a mixture of egg and plaque salts (the weight ratio of egg and plaque salts was 1:20, EC+PBS) was divided into dose groups by concentration. On the 49th day of Freund's complete adjuvant administration, all experimental animals were anesthetized after CO ² anesthesia, blood was collected from the abdominal aorta and joint tissue was collected. Blood was centrifuged at 3,000 rpm for 20 minutes to separate serum and used for analysis, and stored at -70°C until analysis.

2) Arthritis severity evaluation

The arthritis index measurement method (rheumatoid scoring system) was used to observe the clinical animal arthritis severity (clinical severity) from 21 to 49 days twice/week interval. If there is no evidence of arthritis, such as edema, redness, or swelling, 0 points, redness with one or two toe swelling, or minimal swelling, with mild edema and redness limited to the ankle joint or ankle. 1 point, positive flushing or local upper extremity swelling, and inability to use the foot freely, 2 cases of severe edema and redness across the ankle joint to the foot muscles, and significant edema and swelling up to the knee. The average value of the measured values of the four feet was calculated with three points of edema and redness from the ankle to the entire toe without using the feet freely. The results are shown in FIG. 6.

After all the experimental animals were sacrificed on the 49th day of the experiment, the metatarsal distal portion was extracted from the distal tibia to include the right ankle joint, fixed in 4% neutral formalin solution at 4℃ for 24 hours, and then in formic acid. Immersion was immersed for 5 days, and then demineralized. Paraffin blocks were fabricated through general tissue processing using demineralized tissue. Hematoxylin & eosin (H&E) staining was performed after cutting to a thickness of 5 μm using a rotary microtome. The results are shown in FIG. 7.

As shown in Figs. 6 and 7, the mixture of egg yolk and bamboo salt (EC+PBS) in an animal model of rheumatoid arthritis induced by collagen was administered for 4 weeks. These results were the same as the general symptoms of the collagen-induced animal model. In the Dexamethasone-administered group used as a positive control drug, after a weak edema was observed in the early stage of observation, the reaction disappeared from the observation day on the 31st day, and a clear effect could be confirmed. In the group administered with the mixture of egg yolk plaque and bamboo salt (EC+PBS), the dose-dependent response was shown at the initial stage of administration, showing a more pronounced result in the 400 mg/kg administration group, but a similar effect was observed in the observation at day 49. These results were found to be statistically significant compared to the CIA-control group.

3) Hematological examination

The concentration of the anti-collagen antibody in the serum was measured by an immunoenzyme reaction method (ELISA). A standard concentration curve was prepared based on the measured value of the standard collagen solution, and the concentration in the serum was calculated by substituting the absorbance of each sample. The results are shown in FIG. 8.

As shown in Fig. 8, a mixture of egg yolk and adenitis for collagen antigen-specific antibody anti-collagen anti-body (anti-type II collagen mAb) produced in collagen-induced rheumatoid arthritis animal model (EC+PBS) ), the anti-collagen antibody concentration in the blood was confirmed by ELISA. As a result, 2.34 ± 0.54 units/ml in the normal control group, 31.2±4.16 units/ml in the CIA control group, and 200 mg/kg bw in the test substance administration group (EC+PBS 200) 29.48±3.11 units/ml, 400 mg/kg bw The administration group (EC+PBS 400) was measured at 24.36±4.32 units/ml, and it was confirmed that the increase of the anti-collagen anti-body of the mixture of egg yolk and autism was significantly reduced. In the positive control drug dexamethasone administration group, it was markedly reduced to 1.94±0.34 units/ml compared to the CIA control group.

Simple modifications or changes of the present invention can be easily carried out by those skilled in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

Claims (4)

A pharmaceutical composition for preventing or treating arthritis, comprising a mixture of egg yolk and bamboo salt as an active ingredient. According to claim 1, In the mixture, the egg yolk: bamboo salt is contained in a weight ratio of 1:1 to 1:60, a pharmaceutical composition for the prevention or treatment of arthritis. According to claim 1, The arthritis will include one or more of degenerative arthritis, rheumatoid arthritis, pharmaceutical composition for preventing or treating arthritis. The pharmaceutical composition for preventing or treating arthritis according to claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

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