KR100486791B1 - Pharmaceutical composition for treating rheumatoid arthritis containing hyssop or milky skin extract - Google Patents

Abstract

The present invention not only inhibits the production of superoxide, prostaglandin (PGE ₂ ) and interleukin-1β for the treatment of arthritis, but also degrades collagen protein, which is a substrate of connective tissue, to collagenase (Collagenase). The present invention relates to a pharmaceutical composition for treating rheumatoid arthritis, containing urine extract, milk extract, or a mixture thereof, which inhibits the activity of the enzyme and promotes collagen protein synthesis.

Description

Pharmaceutical composition for treatment of rheumatoid arthritis containing milk extract or milky skin extract

The present invention relates to a pharmaceutical composition for treating rheumatoid arthritis containing milk extract or milky skin extract. More specifically, the present invention not only inhibits the production of superoxide, prostaglandin (PGE ₂ ), interleukin-1β (Interleukin-1β) for the treatment of arthritis, but also degrades collagen protein, which is a substrate of connective tissue. The present invention relates to a pharmaceutical composition for treating rheumatoid arthritis, which contains a sesame extract, a milk extract, or a mixture thereof which inhibits the activity of the enzyme and simultaneously promotes collagen protein synthesis.

Rheumatoid arthritis is reported to be caused by various causes of symmetric chronic synovialitis. Especially, it is considered to have a genetic background because it is related to HLA-Dr4 among leukocyte antigens. It is thought that the occurrence may be related, but there is no direct evidence for this.

Microorganisms such as Epstein-Barr Virus have been reported to be associated with the disease, but this is also not considered to be one of the causes of multifactorial diseases. The initial arterial synovial lesions for any cause were the findings of intravascular synovial microvessels and the synovial cells. The initial arterial synovial lesions were the findings of intravascular synovial damage and proliferation of synovial cells. It is not known what cells initially caused these lesions, but over time, a characteristic histologic finding, called Panus, appears. This panus is histologically composed of synovial cells, lymphocytes, plasma cells, mast cells and macrophage, and the various cytokines and collagenase secreted by them. Enzymes such as collagenase destroy joints and periarticular tissues. Rheumatoid factor is secreted from the plasma cells of Panus, in particular by forming an immunocomplex with lgG immunoglobulin to activate the complement and various lysogenic pros by phagocytosis by neutral polynuclear leukocytes and macrophages. Lysosomal protease and oxygen radicals are produced. In addition, activation of lipooxygenase and cyclooxygenase metabolic pathways is also involved in the destruction of joint tissue. In addition to these changes, vasculitis may appear in severe rheumatoid arthritis, and thus rheumatoid arthritis may be considered as a local joint disease and a progressive autoimmune disease.

The medical drug treatment for treating rheumatoid arthritis can be roughly classified into three types. First, it is divided into first-level drugs used to improve clinical symptoms only, second, disease modifying drugs used for the purpose of preventing or delaying disease progression, and third, immunosuppressants or cytotoxic agents. The first category includes aspirin, nonsteroidal anti-inflammatory drugs, simple analgesics and less than 7.5 mg of glucocorticoids per day. The most widely used nonsteroidal anti-inflammatory agents are indomethacin, naproxen, piroxicam, sulindac, ibuprofen, tolmetin, diclofenac, etc. There is no evidence that the efficacy of these drugs is superior to aspirin, but the gastrointestinal disorders are only mild. Reduction of renal function, elevated liver enzymes, and side effects of skin rashes can occur as with aspirin. You must lose. Drugs belonging to Group 2 are almost empirically discovered drugs such as forbidden drugs, D-penicillamine, antimalarial drug and sulfasalazine. Since several weeks or weeks must pass, anti-inflammatory drugs must be accompanied. It is certain to reduce inflammation and delay disease progression, but the true meaning has not been proven to date. Each drug is toxic and requires close observation.

Glucocorticoids have excellent anti-inflammatory effects, but they should be avoided if possible due to various side effects. When not controlled by the two-stage drugs, immunosuppressants or cytotoxic agents such as azathioprine or cytoxan may be used. In addition, methotrexate, an anti-antagonist of folic acid, has recently been in the spotlight, but the effect is rapid, but there are some side effects of cirrhosis and lung fibrosis, which should be used with caution.

In order to solve the problem of these side effects and to maximize the efficacy in the systemic or local, the absorption of the drug through the skin using a patch that is a transdermal drug is being performed. In such transdermal administration, pH in the gastrointestinal tract, residence time in the gastrointestinal tract, changes in gastrointestinal motility, changes in the gastrointestinal tract, bacteria, food, etc., and degradation by enzymes in the gastrointestinal tract can be avoided. Since circulating in the body does not pass through the liver first, the drug efficacy is not greatly reduced by metabolism in the liver, and it is convenient to discontinue the medication especially when the patient's condition changes or side effects appear by administering transdermal.

In consideration of these advantages, the study on the transdermal drug delivery system has recently increased, and US Patent Nos. 4393076 and 4534980 reported that the propionic acid nonsteroidal anti-inflammatory drugs were used as ointments or creams as external preparations to treat the rheumatoid arthritis. In the product using the patch, commercial products such as estradiol as a motion sickness treatment, chronidine as an antihypertensive drug, nitroglycerin as an angina drug, and ketoprofen as an arthritis drug are prepared, and the drug is controlled for a short time. It is also possible to reduce the number of side effects of the drug by reducing the side effects that are released in a large amount to be absorbed by the living body, and to maintain a constant blood concentration of the drug over a long time.

In an attempt to develop a patch to increase the absolute amount of the drug in order to ensure that the drug is absorbed into the percutaneous by using a patch, US Patent No. 4031894 discloses that the drug is more than saturated in a base such as a patch, ointment, cream. If the drug is dispersed in the form of fine particles of recrystallization and applied to the skin surface, the drug is gradually dissolved and absorbed through the skin, but it is difficult to re-dissolve and the absorption rate is not so high.

As another method, US Pat. No. 4,814,168 can contain the maximum amount of drug in the patch, and it is a transdermal administration system that can maintain the adhesive properties of shear, adhesive force, and peeling properties, and consists of drug, vinyl acetate and ethylene monomer. It contains polypolymers, rubbers and adhesives.

US Pat. No. 4,814,168 contains hydrophilic active ingredients in hydrophobic polymers for the transdermal administration of active ingredients that are difficult to absorb percutaneously, and water is used as a solvent, which is suitable for transdermal administration of hydrophilic drugs. Since it is not suitable for transdermal administration of oil-soluble drugs, Korean Patent Publication No. 9513448 is suitable for transdermal administration of oil-soluble drugs, and it is possible to maintain a long-term efficacy by using a patch containing a pressure-sensitive adhesive having a multi-layered structure having different functionalities in each layer. Although there are reports, it is not suitable as a patch that can be transdermally administered when water-soluble and oil-soluble active ingredients such as plant extracts are mixed.

Accordingly, the present inventors conducted extensive research on herbal extracts to search for medicinal substances that are potent in rheumatoid arthritis and did not cause allergic reactions with the skin, and confirm that the dew and milky skin extracts are excellent in efficacy. In order to develop a patch that can arbitrarily control the release properties of water-soluble and oil-soluble active ingredients over time using extracts, drug storage layer is prevented and drug release is controlled through membrane filter paper. Afterwards, a method of designing transdermal absorption through a thin adhesive layer and adding a substance that has a drug transport control function and a transdermal absorption promotion function to the solid to enable release over time of the drug enables sustained effective blood concentration. To develop a method to control patients with rheumatoid arthritis In conclusion, clinical trials revealed excellent efficacy and completed the present invention.

The present invention relates to a pharmaceutical composition for treating rheumatoid arthritis, characterized in that it contains hyssop extract, milky skin extract or a mixture thereof as an active ingredient.

Drugs containing wadded and milky skin extracts according to the present invention can be prepared in any form for the treatment of rheumatoid arthritis. Such forms can be prepared, for example, in tablets, capsules, powders, ointments, solutions, gels, pastes, patching agents, granules.

One of the effective herbal medicines in the pharmaceutical composition of the present invention is dried in the sun after removing the beard roots by digging roots of Achyranthes japonica, which is a plant and amaranth distributed in all parts of Korea, Japan and South Hwanghai Province in China. It is yellowish or yellowish brown stick or slightly bent length of 15-90cm and 3-7cm in diameter, and there are many vertical wrinkles and marks of side roots in various places. The plane is grayish white or light brown, and the neck of the center is yellowish white, and the quality is hard but brittle. It is almost odorless and tastes slightly sweet and mucus. Under the microscope, the skin and the neck are distinguished by a clear formation layer. At the center of the neck, there is a small protozoa, which surrounds it, and the concentric annulus is arranged outward. In the flow cell, it contains the matter of calcium hydroxide, and starch granules are not seen. Its pharmacological action is known to be effective in diuretic, painful, arthritis, etc. The main ingredients are saponin, oleanolic glycoside, insect hormonal hormone (inokosterone, ecdysterone), and betain hydrate. Etc.)

Yubaekpi, another effective herb used in the pharmaceutical composition of the present invention, is an elmaceae plant, which is distributed in central North Korea, China, and Japan, respectively, Ulmus macrocarpa, Ulmus pumila, and elm. davidiana) and the roots of the stem or root are collected and dried in the sun. Its pharmacological action is known to have diarrhea, small seedlings, canned foods, species, and single action. The main ingredients are beta sitosterol ( β- sitosterol), plants It consists of sterol and tannin.

In the present invention, water and alcohol extracts are used as extracts of dew and milky skin. The alcohol extract of dew and milk milk skin used in the present invention is obtained by drying the dried milk and milk milk skin, crushing and then pulverizing, extracting with alcohol and filtering and concentrating the filtrate under reduced pressure. Alcohol extraction solvents that can be used for extraction of dew and milky skin include lower alcohol solvents having 1 to 4 carbon atoms such as ethanol, methanol, propanol and butanol, and preferably ethanol.

In the pharmaceutical composition of the present invention, it is appropriate to use the alcohol extracts of the dew and milky skin at a ratio of 0.001 to 10% by weight, preferably 0.01 to 5% by weight, respectively, based on the total weight of the composition. According to the present invention, even in the case of mixing and mixing the extract of the dew and milky skin, it is preferable that each extract is present in the same ratio as described above. When the content of each herbal extract is less than 0.001% by weight, it is not suitable because the efficacy and effect of leaning against rheumatoid arthritis cannot be expected, and when the content of the herbal extract exceeds 10% by weight, the stability of the product may be lowered.

The pharmaceutical composition of the present invention is also composed of three elements, a support, a solid, and a liner, which are commonly used in the preparation of a patch, and a polyethylene, polypropylene, nonwoven fabric, cotton cloth, etc. are used as a support, and a silicone or fluorine is used as a liner. A release film containing a release agent is used, and the solid part is composed of an active ingredient, a pressure-sensitive adhesive, a humectant, an inorganic filler, a buffer, and a preservative, and may be manufactured by the following method.

Mixes and stirs dew and milk extracts as an active ingredient, sodium polyacrylate, sodium carboxymethylcellulose, gelatin, etc. as a tackifier, glycerin as a humectant, zincification as an inorganic filler, kaolin, etc., and emulsifiers, preservatives, and flavoring agents. Manufactured by application, cutting to each product size and packaging.

More specifically, the adhesive portion used in the present invention includes sodium alginate, gelatin, casein, semisynthetic polymer, methyl cellulose, ethyl cellulose, ethyl cellulose, and the like. Hydroxycellulose, Carboxymethyl Cellulose, Dextrin, Carboxymethyl Starch, Synthetic Polymer Polyacrylic Acid, Polyvinyl Alcohol, Methoxy Ethylene (methoxyethylene), polyvinyl ether (polyvinyl ether) is used individually or in combination of 1 to 40% by weight, and wetting agents are used to maintain the state of the patch and prevent drying. Such wetting agents are glycerin, sorbitol liquid, polyethylene Glycol and propylene glycol, poloxamer 407, monoglyceride (Myverol 18-99), 5-30% by weight, alone or in combination of two or more The dragon. In particular, polyethylene glycol 200, map polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000 can be used. As the inorganic filler, 0.1 to 5% by weight of kaolin, bentonite, zinc oxide, zinc oxide, and titanium dioxide are used alone or in combination of two or more thereof, and acetaldehyde is used as a crosslinking agent. , Glutaraldehyde (glutaraldehyde), glyoxal (glyoxal), dialdehyde starch (dialdehyde starch), dimethyl ketone (dimethyl ketone) alone or in combination of two or more kinds to use 0.01 to 5% by weight. Buffers that adjust the pH of the patch include, but are not limited to, alkali metal salts of phosphorous acid, particularly sodium phosphate dibasic, sodium diphosphate dibasic, sodium triphosphate, citric acid and sodium citrate, phosphoric acid, hydrochloric acid, sodium hydroxide and sodium pyrophosphate and pyrophosphate. However, the buffer used in the solid composition of the present invention was adjusted to a pH of 5 to 8.0 by appropriately mixing two kinds of sodium phosphate, sodium phosphate, and sodium phosphate. In order to prevent contamination of microorganisms that may occur during the preparation and use of the patch, paraoxybenzoic acid methyl, paraoxybenzoic acid propyl, benzoic acid, sodium benzoate, salicylic acid, etc., which are generally licensed for use in foods and pharmaceuticals To 0.01 to 0.5% by weight.

When using the pharmaceutical composition of the present invention clinically, the patch prepared in the ratio as described above may be attached to the affected area with rheumatoid arthritis once every two days, but may be appropriately increased or decreased depending on the progress of the condition.

Hereinafter, the present invention will be described in more detail based on Examples and Comparative Examples, but the technical scope of the present invention is not limited in any way by these.

Preparation Example 1

Take 100 g of the powder obtained by pulverizing the dried dew powder, add 1000 ml of 95% ethanol, deposit it for 3 days, filter it using No. 1 Whatman No. 1, and concentrate under reduced pressure to dry the dew powder. 30 g of extract was obtained.

Preparation Example 2

100 g of the powder obtained by pulverizing the dried milky skin was taken, and 1000 ml of 95% ethanol was added thereto, followed by immersion extraction for 3 days, filtered using No. 1 Whatman No. 1, and concentrated under reduced pressure to dry. 40 g of dew extract was obtained.

Examples 1-18 and Comparative Examples 1-18

The patch according to the present invention after mixing and stirring, spreading all over, cutting and packing to the size of each product according to the conventional patch preparation method according to the ingredient combination ratio shown in Tables 1 to 9 (Examples 1 to 18) And a comparative patch (Comparative Examples 1 to 18) were prepared.

Experimental Example 1. Inhibitory effect on the superoxide generation of (Examples 1 to 18) of the adhesive agent of the present invention

Intravenous blood collected using citric acid as an anticoagulant from a healthy adult without centrifugation was centrifuged at 1200 rpm for 10 minutes, and then the leukocyte concentrate was recovered first, and then diluted in a ratio of 1: 1 with RPMI 1640 medium. After 12 ml of Ficoll-Paque was added to a 50 ml centrifuge tube, 30 ml of diluted blood was carefully added to form a middle layer, followed by centrifugation at 1600 rpm for 30 minutes, and then the upper layer containing serum was removed. The contained intermediate layer was carefully extracted with a sterile pipette into a new centrifuge tube, then 3 times RPMI 1640 medium was added, centrifuged at 800 rpm for 10 minutes, discarded the supernatant, 10 mL RPMI 1640 medium was added and gently pipetting After centrifugation at 800 rpm for 10 minutes, the supernatant was discarded and pipetting with the addition of HBSS (Hanks' Blanced Salt Solution) buffer solution. 0.45 mL of human mononuclear wall blood cells were dispensed into 24-well plates at 10 ⁶ cell / well and incubated for 2 hours in aseptic conditions under 95% air, 5% CO ₂ , and 100% humidity conditions, followed by FMLP (N-Formyl). -Met-Leu-Phe) was treated with 0.05 mL of 10 ^-6 M and incubated at 37 ° C for 15 minutes to stimulate the cells, followed by 80 μM of 0.1 mL cytochrome C and 30 μg 0.1 mL superoxide disc. 0.1 ml of the adhesive part of the patch of the examples and comparative examples diluted three times with superoxide dismutase and HBSS was added, and HBSS (Hanks' Balanced Salt Solution) was added so that the remaining total reaction solution was 0.9 ml. Insulate for 10 minutes at 0.1 ° C and add 0.1 ml of stimulated Zymosan A to a final concentration of 1.3 mg / ml, insulate for 90 minutes at 37 ° C with shaking, and place it in a refrigerator at 4 ° C for 10 minutes. After stopping the reaction, centrifuged at 4 ℃, 1500rpm, 10min, and the supernatant was optical at 550nm. The density (OP) was measured and the amount of superoxide anion produced was calculated by the following equation.

ΔO.D. = (B-D)-(A-C) = (B + C)-(D + A)

In the above test results, in Example 1, 5, 7, 9 and 11 containing more than 0.001% of the dew was found that the inhibitory effect of superoxide production increased with increasing concentration of the dew, while containing milky skin Examples 2, 4, 6, 8, 10 and 12 were found to have no superoxide inhibitory effect. Examples 13, 14, 15, 16, 17 and 18, which contained both dew and milky skin, also showed that the inhibitory effect on superoxide production increased with the concentration of dew.

Experimental Example 2. Inhibitory effect on the activity of collagenase enzyme which is a defect tissue degrading enzyme of the adhesive agent of the present invention (Examples 1 to 18)

The results of the inhibitory effect on the collagenase, a connective tissue degrading enzyme, with the adhesive patch were as follows.

100 μl of 2% red collagen Azocoll solution was added to 25 1.5 ml Eppendorf tubes, one of which was used as a blank, and three tubes were purchased from Sigma. One collagenase type I standard enzyme solution (collagen degrading activity: 315 units / mg) was added to 10, 100, 200 ppm, and 100 μl of collagenase enzyme was added. Each of the experimental group adhesive adhesive (Examples 1 to 18) and the comparative group adhesive adhesive (Comparative Examples 1 to 18) were mixed with distilled water in a ratio of 1: 2 and completely homogenized, followed by centrifugation at 5000 g for 10 minutes. After adding 10 µl of the supernatant, 500 µl of the buffer solution (0.05 M Tris-Hcl, 1 nM CaCl ₂ , 7.8) was added to the reaction mixture for 18 hours at 37 ° C. incubator for 5 minutes at 10000 g for 5 minutes. Coke not broken down by centrifugation Xen takes a supernatant solution containing precipitated and decomposed collagen, measures absorbance at 540 nm, prepares a standard activity curve, converts the enzyme activity concentration from the standard curve, and compares and evaluates the enzyme activity of the experimental and control groups as follows. Got.

(Unit:% = enzyme activity of test group x 100 ÷ enzyme activity of control group)

(Unit:% = enzyme activity of test group x 100 ÷ enzyme activity of control group)

In the above test results, the inhibitory effect on collagenase activity in Examples 1, 5, 7, 9 and 11 containing 0.01% or more of the dew was increased as the concentration of the dew was increased. In Examples 2, 4, 6, 8, 10, and 12, the inhibitory effect on collagenase activity was increased with the increase of the milky skin concentration, and both the dew and milky skin had excellent inhibitory effect on the collagenase activity. Example 13, 14, 15, 16, 17 and 18, which contained both dew and milky skin, showed synergistic effect.

Experimental Example 3. Inhibitory effect on the production of interleukin (IL-1β) of mononuclear leukocytes of the adhesive agent of the present invention (Examples 1 to 16)

Blood mononuclear leukocytes isolated from blood were added to 0.8 wells in a 24-well plate and divided into 10 ⁶ cell / well, and 200 μl of RPMI 1640 medium was added to the control, 100 μl of E. coli LPS (250 ppm). And wells containing 100 μl of LPS (250 ppm) and 100 μl of adhesive patch diluted three-fold with RPMI 1640 medium as an experimental group, and incubated for 24 hours, and then 50 μl of arachidonic acid was added thereto. Incubate for more minutes. Interleukins (IL-1β) in a well of the antibody is attached to a 96-well freight (96-well plate), IL -1 β 50㎕ of the standard solution (0, 10.24, 25.6, 64 , 160, 400pg / well) of 50 μl of the above cell culture solution was added to the experimental group wells, and 50 μl of Biotinylated Antibody Reagent was added to all the wells, and then maintained at 25 ° C. for 3 hours, followed by 0.01 buffer containing 0.05 wt% of Tween 20. M phosphate buffer solution, pH 7.5), washed three times, 100 μl Streptavidin-HRP Conjugate was added to all wells and maintained at 25 ° C. for 30 minutes, and then washed three times with wash buffer solution and 100 μl of enzyme substrate. Immediately add and keep the plate open in the dark at 25 ° C for 30 minutes, add 100 μl of 0.18M sulfuric acid, and measure the absorbance at 450 nm using a microplate reader. Calculate the amount of interleukin produced in the experimental group.

The efficacy and effects of IL-1 β production on human mononuclear leukocytes stimulated with E. coli LPS were tested to show that the inhibitory effect was 3, 5, 7, 9, and 11 containing more than 0.01% concentration of hyssop extract. in examples 2, 4, 6, 8, 10 and 12 containing the milky blooming, while shown to be elevated as the concentration of wooseul increase was no generation inhibitory effect of IL-1 β. Examples 13, 14, 15, 16, 17, and 18 containing both dew and milky skin also showed that IL-1 β production inhibitory effect increased with the concentration of dew.

Experimental Example 4 Prostaglandin (PGE) of mononuclear leukocytes of the adhesive agent of the present invention (Examples 1 to 18) ₂ Inhibitory effect on production

The experimental results of the inhibitory effect on the production of prostaglandins, which is a gingival inflammation-causing substance, using the adhesive patch are as follows.

In this test method, the production of prostaglandins (PGE ₂ ) induced by stimulating human mononuclear leukocytes with Lipopolysaccharide, an E. coli cell wall component, was carried out by the immuno-diagnostic method of antigen-antibody. Examples 1 to 18) and comparative group ointment impregnated ointment (Comparative Examples 1 to 18) were mixed with the medium at a ratio of 1: 2, completely homogenized, and centrifuged at 5000 g for 10 minutes to compare the inhibitory effect. Evaluated. Specific test methods contained 50 μl buffer solution (0.9% NaCl, 0.1% bovine serum albumin, 0.5% Kathon) in a blank well of a 96-well plate containing 1 gG of goat anti-mouse. 0.1 M phosphate buffer solution) was added to the standard wells (0, 2.5, 5, 10, 20, 40, 80, 160, 320 pg) and 50 μl of PGE ₂ standard solution was added to the well. 50 μl of the experimental group patch adhesive (Examples 1 to 18) and the comparative group patch adhesive (Comparative Examples 1 to 18) were added to the wells set in the Example and Comparative Examples, and 50 μl of PGE ₂ was added to the wells. The antibody was added to all wells except the blankwell, then 50 μl of PGE ₂ conjugate peroxidase was added to all wells except the blankwell, covered with 96 wells-plate and for 1 hour at 25 ° C. After maintaining, wash 4 times with washing buffer solution (phosphate buffer solution containing 0.05% Tween 20: pH 7.5) and at room temperature 150 μl of enzyme substrate (3, 3 ', 5, 5'-tetramethylbenzidine / hydrogen peroxide dissolved in 20% dimethylformamide) was added immediately, held at 25 ° C. for 30 minutes, and 100 μl of 1M sulfuric acid was added. After measuring the absorbance at 450 nm with a microplate reader to prepare a standard curve by the absorbance value of the standard solution to calculate the amount of prostaglandin produced in the experimental group.

The efficacy and effects of PGE ₂ production in human monocytic leukocytes stimulated with E. coli LPS were tested and the inhibitory effects of 3, 5, 7, 9, and 11 containing hyssop extract increased as the concentration of hyssop On the other hand, in Example 2, 4, 6, 8, 10 and 12 containing milky skin, there was no inhibitory effect on PGE ₂ production. Examples 13, 14, 15, 16, 17, and 18, which contained both dew and milky skin, also showed that the inhibitory effect of PGE ₂ production increased with the concentration of dew.

Experimental Example 5. Effect on the production of collagen protein of human fibroblasts of the adhesive agent of the present invention (Examples 1 to 18)

1mL of DMEM medium containing 10% FBS was dispensed into each well so that primary cultured fibroblasts were 10 ⁶ cells / well in a 24-well plate, and cultured for one day. After incubation, the cell layer attached to the bottom was washed with HBSS buffer solution, and then 0.8ml of MEM medium containing no serum and Proline was added, and then the experimental group adhesive group (Examples 1 to 18) and the comparative group adhesive group ( Comparative Examples 1 to 18) were mixed with the medium at a ratio of 1: 2, completely homogenized, centrifuged at 5000 g for 10 minutes, and 100 µl of the supernatant was added thereto, followed immediately by a culture solution containing 100 µl of ¹⁴ C-Proline (10 µ Ci). The cells were cultured with. After 24 hours the collagen protein was measured. First, in order to measure the amount of extracellular total protein, the culture medium of each well was placed in a sealed dialysis tube at one end and sealed at the other end. Cold Buffer (Tris-Hcl 0.05mol / L, NaCl 0.2mol / L, CaCl ₂ Dialysis was completed with 0.05mol / L and phenylmethylsulfonylfluoride 0.3 mN) for 24 hours, and then 100 μl of each was added to the counting vial and 10 ml of scintillation coctail was added to the liquid scintillation counter (LSC) for 1 minute. Measured. In order to measure the total amount of intracellular total protein, 0.1N NaOH and 0.3mN of phenylmethylsulfonylfluoride were added to each well from which the cell culture medium was removed, and then maintained at 60 ° C. for 30 minutes to destroy cell membranes. Put one end in a sealed dialysis tube and seal the other end with cold buffer (Tris-Hcl 0.05mol / L, NaCl 0.2mol / L, CaCl ₂ 0.05mol / L, phenylmethylsulfonylfluoride 0.3mN). After completion of time dialysis, 100 μl of each was added to the conunting vial, and 10 μl of scintillation coctail was added. In order to measure the total amount of intracellular and extracellular total collagen protein, 100 μl of dialysis-treated cell culture and cell homogenate were taken and placed in 1.5ml microtube, followed by collagenase buffer (0.05M Tris-Hcl, 1mM CaCl ₂ , 0.3mM phenyl Methylsulfonylfluoride) and 100 μl of collagenase enzyme (100 ppm), and then maintained at 37 ° C. for 3 hours to completely decompose collagen and then remove 50% trichloroacetic acid and 500 µl of a solution containing 1% tannic acid was added, precipitated at 4 ° C for 30 minutes, centrifuged at 1000xg for 5 minutes, 100 µl of the supernatant was added to the counting vial, and 10 ml of scintillation coctail was added to the LSC. Radioactivity was measured by 1 minute.

In order to study the efficacy and effect on the collagen synthesis of human fibroblasts, the present invention was treated with a cell culture medium containing ¹⁴ C-Proline as described above. Examples 2, 4, 6 containing milky skin extracts , 8, 10 and 12 showed that the collagen synthesis promoting effect was increased as the concentration of milky skin increased, whereas in Examples 3, 5, 7, 9 and 11 containing dew, there was no collagen synthesis promoting effect. appear. Examples 13, 14, 15, 16, 17 and 18, which contained both dew and milky skin, were also shown to increase the collagen synthesis promoting effect according to the concentration of milky skin.

Experimental Example 6. Animal experiments on the efficacy and effects of the present invention patch (Examples 1 to 8)

The clinical experimental method of the present invention patch was performed in the following manner.

Wistar male rats weighing 200-250 g were divided into experimental group and light anesthetized with ether, and then injected into Camplete Freynd's adijuvant on the right rear sole. The group injected with only liquid paraffin was used as a control. From 5 days to 19 days after the injection, the experimental group patch (Examples 1 to 8) and the comparative group patch were applied to the right hind paw of the experimental group at the same time at two days intervals. The following results were obtained as% with the control group.

In the above clinical trials, it was found that IL-1 β and prostaglandin-induced gingivitis, superoxide and collagen synthesis, which inhibit the enzyme activity of collagenase, which degrades periodontal tissue, promote collagen and The inhibitory effect of foot edema development of the inventive patch (Examples 1 to 8) containing milky skin extracts capable of inhibiting the enzymatic activity of the agent was compared with no dew or milky skin from 1 week to 1 month. It was shown that the efficacy is superior to the examples (1 to 8), the foot edema of the comparative example is continuously increased with time after 5 days, while foot edema when using the patch developed by the present invention Was suppressed, and foot edema was significantly reduced over time. Examples 3, 4, 5, 6, 7, 8 containing both dew and milky skin Documentation showed that a synergistic effect on the anti-arthritic activity.

1 shows a cross-sectional view of a patch according to the present invention.

* Explanation of symbols for main parts of the drawings

1: support 2: adhesive 3: release film

Claims (5)

Rheumatoid arthritis treatment composition comprising milk extract as an active ingredient. According to claim 1, wherein the composition is rheumatoid arthritis treatment composition comprising 0.001 to 10% by weight of the milky skin extract. According to claim 1, wherein the composition is rheumatoid arthritis treatment composition comprising 0.01 to 5% by weight of milk extract. The composition for treating rheumatoid arthritis according to claim 1, wherein the formulation of the composition is a tablet, capsule, powder, ointment, solution, gel, paste, patch or granule. The composition for treating rheumatoid arthritis according to claim 1, wherein the milk extract is extracted with a solvent selected from the group consisting of water, methanol, ethanol, propanol and butanol.

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