KR100595735B1 - Herbal pharmaceutical composition for regenerative agent of cartilaginous tissue and treatment of osteoarthritis - Google Patents

Abstract

The present invention relates to a herbal composition for treating cartilage regeneration and osteoarthritis, and more particularly, to a composition for treating cartilage regeneration and osteoarthritis including vigor, jinjin and lobules.

The composition of the present invention has an anti-inflammatory effect, anti-inflammatory analgesic effect, inhibitory effect on articular tissue degrading enzymes, inhibitory effect on inflammation-induced enzymes, inhibition of foot edema, inhibition of increase in joint synovial fluid, inhibition of total protein increase in joint synovial fluid It can be used to treat cartilage regeneration and osteoarthritis.

Active extract, human extract, leaflet extract, proteoglycan, prostaglandin, MMP-9, COX-2, cartilage regenerant, osteoarthritis.

Description

Herbal composition for cartilage regeneration and osteoarthritis treatment {HERBAL PHARMACEUTICAL COMPOSITION FOR REGENERATIVE AGENT OF CARTILAGINOUS TISSUE AND TREATMENT OF OSTEOARTHRITIS}

Figure 1 shows the anti-inflammatory effect of individual extracts of the active, phosphorus and lobular and herbal compositions of Examples 1-1 to 1-4, 2 and 3 for osteoarthritis induced by LPS.

Figure 2a to 2c shows the degree of carbon monoxide production after treatment of the herbal compositions of Examples 1-4, 2 and 3 to 0.8, 4, 20 ㎍ / ㎖ in LPS-induced mouse macrophage in Experimental Example 2 LPS represents the total amount of nitric oxide produced in the control group after induction of inflammation.

3A to 3B show prostaglandin E ₂ generated after treatment of the herbal compositions of Examples 1-4, 2, and 3 with 0.8, 4, and 20 μg / ml of mouse macrophage induced by LCM in Experimental Example 3 It shows the amount of. CM shows the total amount of prostaglandin E ₂ of the control group produced after osteoarthritis induction by LCM.

Figures 4a to 4c is generated after treating the herbal composition of Examples 1-4, 2, and 3 to 0.8, 4, 20 ㎍ / ㎖ in rabbit cartilage tissue cells of osteoarthritis induced by LCM in Experimental Example 4 It shows the total amount of nitrogen monoxide. CM represents the total amount of nitric oxide in the control group produced after osteoarthritis induction by LCM.

Figures 5a to 5c is produced after treating the herbal composition of Examples 1-4, 2, and 3 with 0.8, 4, 20 ㎍ / ㎖ in rabbit cartilage tissue cells of osteoarthritis induced by LCM in Experimental Example 5 The total amount of prostaglandin E ₂ is shown. CM shows the total amount of prostaglandin E ₂ of the control group produced after osteoarthritis induction by LCM.

Figures 6a to 6c shows the results of the degradation of proteoglycans after treating the herbal compositions of Examples 1-4, 2, and 3 with 0.8, 4, 20 μg / ml of osteoarthritis induced by LCM in Experimental Example 6 The total amount is shown. CM shows the total amount of proteoglycans of the control group produced after osteoarthritis induction by LCM.

FIG. 7 shows the activated MMP-9 after treating the herbal compositions of Examples 1-4 and 2 with 0.8, 4, and 20 μg / ml of osteoarthritis induced by LCM in Experimental Example 7;

The total amount of MMP-2 is shown. CM represents the total amount of MMP-9 in the control group generated after osteoarthritis induction by LCM.

FIG. 8 shows COX-2 expressed after treating the herbal compositions of Examples 1-4, 2, and 3 with 0.8, 4, and 20 μg / ml of rabbit cartilage tissue cells induced by LCM in Experimental Example 8; It shows the total amount of. CM refers to the total amount of COX-2 expressed after osteoarthritis induction.

Figure 9 shows the degree of inhibition of foot edema in rats of the herbal compositions of Examples 1-4 and 2 in Experimental Example 9.

Figure 10 shows the pain inhibitory effect by the herbal extracts of Examples 1-4 and 2 in Experimental Example 12.

Figure 11 shows the effect of the herbal composition of Example 2 on cartilage regeneration in the animal model induced osteoarthritis.

Figure 12 shows the effect of the herbal composition of Example 2 on the quantitative change of joint synovial fluid in an animal model induced osteoarthritis.

Figure 13 shows the effect of the herbal composition of Example 2 on proteoglycans in the joint synovial fluid in an animal model in which osteoarthritis is induced.

14 shows the effect of the herbal composition of Example 2 on total protein in the synovial synovial fluid in an animal model in which osteoarthritis is induced.

15 shows the effect of the herbal composition of Example 2 on prostaglandin E ₂ in the joint synovial fluid in an animal model in which osteoarthritis is induced.

FIG. 16 shows the herbal composition of Example 2 showing the results of safranin staining and synovial cell distribution of joint tissue in an animal model in which osteoarthritis is induced.

[Industrial use]

The present invention relates to a composition for treating cartilage regeneration and osteoarthritis, and more specifically, anti-inflammatory action, anti-inflammatory analgesic effect, arthrase inhibitory effect, inflammation-related enzyme inhibitory action, foot edema inhibition, inhibiting the increase of joint synovial fluid The present invention relates to a therapeutic agent for cartilage regeneration and osteoarthritis having therapeutic effects such as inhibition of total protein increase in joint synovial fluid.

[Private Technology]

All aerobic organisms, including humans, have self-defense mechanisms against free radical damage caused by oxygen metabolism. However, the production of free radicals beyond the defense of tissues causes many diseases such as arthritis. .

The active oxygen is a singlet oxygen (Superoxide anion, ¹ O _2- ) and hydrogen peroxide (H ₂ O) which is the singlet oxygen produced by the reduction of the triplet oxygen ( ³ O ₂ ), which is the most stable form of oxygen, during the oxidation and reduction process. ₂ ), free radicals such as hydroxy radicals (.OH), which cause various diseases by damaging factors of the immune system such as proteins, DNA, enzymes and T cells.

For this reason, researches on the development of antioxidants have been actively conducted, and antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, vitamin E, and vitamin E, which are natural antioxidants, are preventive antioxidants. C, carotenoids, glutathione and the synthetic antioxidant t-Butyl-4-hydroxyanisole (BHA) dibutyl hydroxy toluene (3,5- (t-Butyl) -4-hydroxytoluene BHT) and many antioxidants are known. However, natural antioxidant enzymes decrease the ability to protect free radicals with aging and synthetic antioxidants cause mutations or toxicity, and thus, there is an urgent need for the development of safe and effective natural antioxidants.

Inflammatory disease refers to the pathological condition of an abscess formed by the invasion of bacteria. Two types of cyclooxygenase (COX), an enzyme that causes inflammation, are known. Of these, COX-1 is involved in the production of prostaglandin (PG) substances in various organs and tissues of the human body, ie, the gastrointestinal tract or kidneys. In comparison, COX-2 is known as an enzyme that acts only at the site of inflammation. Non-steroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, aspirin, and ibuprofen, which are commercially available, simultaneously inhibit COX-1 and COX-2, or mainly COX-1. It inhibits PG, which is essential for maintaining the function of the liver, gastrointestinal tract, or kidney, as well as inflamed tissues, and is known to cause many side effects (Isselbcher et al., Harrison's Principles of Internal Medicine, ( 13th ed) 2, pp 1543-1711).

Osteoarthritis (OA) is the most common disease of the motor system, which accounts for 40 to 60% of all skeletal musculoskeletal diseases, and its prevalence is steadily increasing as the average age increases. 15% of the world's population is diagnosed with OA, and 60% of them are in their 60s or older (Haq I et al., J. Osteroarthritis, 79 (993), pp 377-383).

Arthritis causes severe joint pain due to abrasion of joint cartilage in the joints, leading to severe joint pain. Long-term treatment is required due to the nature of the chronic course of the disease, and complications and side effects from long-term medications occur. Therefore, there is a need for the development of a therapeutic agent that can alleviate pain, maintain and restore the function of articular cartilage and treat complications.

In general, arthritis treatment is focused on eliminating pain by taking simple painkillers in the early stages of pain. However, if pain continues after further progress, anti-inflammatory analgesics with strong anti-inflammatory effects are used. Done. However, such anti-inflammatory drugs will use anti-inflammatory substances of the corticosteroid type (Corticosteroid). For example, long-term use of anti-inflammatory analgesics such as hydrocortisone and betamethasone may cause side effects such as deterioration of gastric, liver and kidney function and regeneration of cartilage cells. As a rule, intraarticular injections should not be given orally.

Therefore, many drugs have been developed that have pain suppression and anti-inflammatory effects and lower the side effects in the most common medications for the treatment of arthritis, and representative examples thereof include diclofenac, aspirin, and ibuprofen. This includes nonsteroidal anti-inflammatory drugs (NSAIDs). However, NSAIDs also have problems with the digestive organs, especially the stomach.

They also have the effect of removing only pain and inflammation without the underlying treatment of arthritis.

The present invention is to solve the above problems, the object of the present invention is the anti-inflammatory action, anti-inflammatory analgesic action, articular tissue degrading enzyme inhibitory action, inflammation-related enzyme inhibitory action, foot edema inhibition, inhibiting the increase of joint synovial fluid To provide a composition for treating cartilage regeneration and osteoarthritis, which has therapeutic effects such as inhibition of total protein increase in joint synovial fluid.

In order to achieve the above object, the present invention provides a composition for treating cartilage regeneration and osteoarthritis, including vigor, jinjin, lobule.

In addition, the present invention extracts the mixture of active, phosphorus and leaflets with distilled water or ethanol aqueous solution, or extracts the active, phosphorus and leaflets with distilled water or ethanol aqueous solution, respectively, to obtain an extract, and concentrate the extract by layer separation with ethyl acetate It provides a method for producing a composition for treating cartilage regeneration and osteoarthritis comprising the step of making.

Hereinafter, the present invention will be described in more detail.

The composition for treating cartilage regeneration and osteoarthritis of the present invention includes active, phosphorus and lobule as active ingredients.

In the present invention, the active ingredient (羌活; Angelica koreana Max.) Is an essential oil component such as limonene (limonene), o-cresol, alpha-pinene (osthenol), bergaptin (bergaptin) It is known to be effective in colds, headaches, neuralgia, etc.).

Injin (茵 蔯, Artemisiae Capillaris Thunb) is a perennial herbaceous deciduous shrub belonging to the genus Asteraceae, Artemisa sacrorum subsp.Kitamura, Artemisa sacrorum subsp.manshuria var. Vestita Kitamura) and the like. In addition, due to its peculiar aroma and medicinal effects, it has brought a lot of attention, and has been used habitually as a edible, medicinal, or herbal medicine. Dongbobom and the treatment and prevention of liver diseases such as liver recovery, fatty liver, liver cancer, hepatitis, jaundice and other diseases in the herbal wood, gastrointestinal disorders such as gastrointestinal disorders, stroke, blood circulation, blood clotting, etc. It is known to be effective in improving circulatory function and treating diabetes. The main ingredients in phosphorus are essential oils, including the glycoside scopoletin, coumarin, oxycarbonic acid, saponin, tannin, and sitosterin resin. ), Such as detoxification, antibacterial, immunity is expected, and the effect of reducing serum lipids are effective in the treatment and prevention of hypertension, diabetes, obesity, stroke and the like.

Leaflet, Perilla sikokiana Nakai ) Is an annual herb of Labiatae. Herbal leaves are called lobules and seeds in Korean herb. The essential oils of all lobules are about 55% of perylaldehyde, 20-30% of l-limonene and α. In addition to a small amount of pinene, it contains arginine, cumic acid, cyanidin-3- (6-p-coumaroyl-β-D-glucoside) -5-β-D-glucoside, The refined oils and fats of the leaves contain isoegoma ketones. Used as a sweat, antitussive, dry, diuretic, soothing and analgesic. Small stems are old stems of the lobules and the roots of the lobules, which are effective in combating gout, sweating and expectoration, lowering the qi, dizziness, body pain and nasal congestion, It is effective in treating runny nose (Information and Shin, Min-Kyo; Doha Herbal Medicine Dictionary, Younglimsa, pp855-856, 1998).

The active is 5 to 50 parts by weight, the phosphorus is 3 to 30 parts by weight, the leaflet is preferably used in 2 to 20 parts by weight. In addition, the composition of the present invention can be used in a composition ratio of 1 to 10: 1 to 6: 1 to 4 active, phosphorus and leaflets. Only in the above content range can be obtained the efficacy of the excellent herbal composition. If it is out of the content range there is a fear that the cytotoxicity from the herbal composition is generated, does not exhibit the desired efficacy, it is not preferable in terms of economics. The herbal composition of the present invention has anti-inflammatory action, anti-inflammatory analgesic action, articular tissue degrading enzyme inhibitory action, inflammation-related enzyme inhibitory action, inhibition of foot edema, inhibition of increase in joint synovial fluid, and the like to treat cartilage regeneration and inflammatory disease. And can be usefully used for prevention. Examples of the inflammatory disease include osteoarthritis, rheumatoid arthritis, shoulder periarthritis, tendonitis, hay salt, peritonitis, myositis and the like. The composition of the present invention has excellent efficacy in treating osteoarthritis of the arthritis.

The extracts from the roots, stems and leaflets may be flowers, leaves, stems, shells, buds, roots, fruits, seeds or plants of plants, preferably the roots are roots, the stems are foliage, and the leaflets are It is a leaf.

The extract may be prepared by pulverizing a plant or a dry matter of a plant, extracting by using an ultrasonic or reflux apparatus using a solvent in a weight ratio of 1: 5 to 1: 10, and then filtering or centrifuging it to obtain a supernatant. have. The solvent may be at least one selected from the group consisting of water, alcohols having 1 to 5 carbon atoms, ethyl acetate, and hexane. It is preferable that the ultrasonic extraction is 2 to 5 hours. The supernatant is fractionated with ethyl acetate (layer separation) to obtain a herbal composition. At this time, the layer separation is preferably carried out three or more times.

The herbal composition may be used in a liquid state, or may be used as a powder from which the solvent is removed by concentration under reduced pressure or lyophilization.

In addition, the herbal composition may be provided in a formulated form having a pharmaceutical effect. Suitable formulations of the herbal composition include, but are not limited to, powders, solutions, pills, tablets, dragees, hard or soft capsules, solutions, suspensions or emulsions, injections, suppositories, and the like. The extract may be prepared in a suitable dosage form using a pharmaceutically inert, organic or inorganic carrier, as a pharmaceutically acceptable salt. That is, when the formulation is a tablet, coated tablets, dragees and hard capsules, lactose, corn starch or derivatives thereof, talc, stearic acid or salts thereof can be used. Also in the case of soft capsule formulations, polyols of vegetable oils, waxes, fats, semisolids and liquids can be used. In the case of solution or syrup form, water, polyols, glycerol, vegetable oils and the like can be used. As suppository carriers, natural or hardened oils, waxes, fats, liquid polyols and the like can be used.

Formulated compositions having a pharmaceutical effect of the present invention may further comprise preservatives, stabilizers, wetting agents, emulsifiers, solubilizers, sweeteners, colorants, osmotic pressure regulators, antioxidants and the like. The method of administration can be readily selected according to the dosage form and can be administered orally or parenterally.

It may also be added to various foods and used in the case of preventing or treating inflammatory diseases and in functional foods for regeneration of cartilage cells. The food to be added may be any food in the market, and is not particularly limited.

In the process of cartilage breakdown that leads to osteoarthritis, some inflammation can be induced, which leads to the release of inflammation-related enzymes, accelerating cartilage damage. In general, excessive amounts of NO are generated at the site of inflammation to promote necrosis of cellular tissues [Moncada, S et al., Pharmacol. Rev. 43: 109-142 (1991). Inhibiting the activity of inducible nitric oxide synthetase (iNOS) that produces these NOs or inhibiting protein expression has become an important key to treatment in many inflammatory diseases. iNOS produces excess NO in a short time in order to defend the living body in response to external stimulation, but in diseases such as arthritis, excessive secreted NO causes secondary side effects such as necrosis and pain. Thus, inhibition of overexpression of iNOS is known to be important for the treatment of osteoarthritis.

In addition, when osteoarthritis is induced, pain increases with intra-articular inflammatory response, which is due to an increase in the concentration of prostaglandin E ₂ , an important factor associated with it in the synovial fluid, thus reducing the concentration of prostaglandin E _{2 in} the synovial fluid. It is also important to improve osteoarthritis.

Proteoglycan (proteoglycan) is composed of protein, sugar, and is a complex molecule that gives elasticity to the cartilage to allow the movement to bend and squeeze by forming a densely packed structure or a frame in the cartilage by intertwining with collagen fibers. It also confines proteoglycans into cartilage tissue to act as a sponge, allowing the cartilage to continue joint movement.

As osteoarthritis progresses, cartilage breaks down along with an inflammatory response, releasing the proteoglycan into the synovial fluid, thereby increasing the amount of synovial fluid and the concentration of proteoglycan in the synovial fluid. Thus, a decrease in proteoglycan concentration in the synovial fluid is an important marker to measure the improvement of osteoarthritis. In addition, when the release of proteoglycans into the synovial fluid is inhibited, proteoglycans are produced in cartilage, which affects the proliferation of chondrocytes, and thus the effect of cartilage regeneration can be measured due to the inhibition of proteoglycan release in the synovial fluid (Ailson M Badger et al., Inhibition of Interleukin-1-induced Proteoglycan Degradation and Nitric Oxide Production in Bovine Articular Cartilage / Chondrocyte Cultures by the Natural Product, Hymenialdisine.The Journal of Pharmacology and Experimental Therapeutics.Vol.290 587-593).

In addition, osteoarthritis-induced cartilage tissue is destroyed by the synthesis and activation of MMP (matrix metalloproteinase), MMP-1 (collagenase-1), MMP-2, MMP-3 (stromelysin-1), MMP-8 (neutrophilcollagenase), MMP-9 (gelatinase), MMP-13 (collagenase-3) and the like increases the expression and activity. Therefore, decreasing the concentration of MMP-9 in synovial fluid is also an important marker to measure the improvement of osteoarthritis.

Cyclooxygenase 2 (COX-2) is an enzyme that synthesizes prostaglandin E ₂ , a pain-causing substance, which inhibits or inhibits the expression of COX-2 because it is promoted by NO and other stimuli. Inhibition is also an important marker for treating osteoarthritis.

In addition, the active oxygen causing inflammation is removed by the antioxidant enzyme SOD (Super Oxide Dismutase, hereinafter referred to as 'SOD'), so the SOD content in the cell is an important marker for improving osteoarthritis.

The herbal composition of the present invention prevents the separation of proteoglycan and prostaglandin E ₂ from chondrocytes, thereby reducing the concentration of the substance in the synovial fluid, inhibiting the production of nitric oxide, the expression of MMP-9 and cyclooxygenase 2 It has been shown to reduce the symptoms of winding by acetic acid. It also reduces paw edema and increases SOD, which serves to remove free radicals.

The herbal composition of the present invention reduces the amount of synovial fluid that increases with the degree of osteoarthritis, reduces the amount of total protein released into the synovial fluid upon cartilage rupture, and reduces the content of proteoglycan in the synovial fluid to prevent the progression of osteoarthritis caused Has an effect.

In addition, osteoarthritis is fundamentally curable as damaged cartilage cells repopulate. The repopulation of such cartilage cells can be judged by observing the surface and distribution of synovial membrane cells by visual observation of articular cartilage and safranin-O staining. In general, in cartilage tissue in which osteoarthritis has occurred, whether or not chondrocytes have proliferated can be determined as the surface of the damaged cartilage tissue becomes smooth. In addition, in safranin-O staining, cartilage tissue is destroyed and the cartilage tissue is not observed in the osteoarthritis-induced cells, whereas the cells in which the cartilage tissue is regenerated are stained in red as compared to the control group. It is possible to determine whether the osteoarthritis is relieved and the chondrocytes proliferate through the degree of formation and the degree of formation of the upper part of the staining site.

In addition, the compositions of the present invention can be administered in various formulations, oral and parenteral, in actual clinical administration. Solid preparations for oral administration include tablets, pills, powders, overgrapes and capsules, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose in a mixed herbal extract. , Lactose and gelatin are mixed and prepared. In addition to simple excipients, glidants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to the commonly used simple diluents, water and liquid paraffin. This may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous and suspension solvents such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and ethyl oleate. Injectable esters such as and the like can be used. Dosage units may contain, for example, 1,2,3 or 4 times the individual dosage or may contain 1/2, 1/3 or 1/4 times. The individual dosage preferably contains an amount in which the effective drug is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dose.

When using the present invention in the form of a pharmaceutical composition, the dose of the active ingredient of the present invention is 1 to 1000 mg / kg, preferably 50 to 300 mg / kg, can be administered 1 to 4 times a day. However, the level of dosage for a particular patient may vary depending on the weight, age, sex, health condition, diet, time of administration, administration method, excretion rate, severity of disease, and the like.

The present invention also provides a food or food additive comprising the active, phosphorus and leaflet extract as an active ingredient.

When the active ingredient of the present invention is used as a food, the extract may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The active ingredient may be included in an amount of 1 to 99.9% by weight, and preferably 10 to 70% by weight, based on the food composition. However, the mixed amount of the active ingredient can be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment), can be used according to the pharmaceutical composition, and is not limited to the embodiments of the present invention. In addition, in the case of long-term intake for the purpose of health and hygiene or for health control, it may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, beverages, tea, and drinks. Alcoholic beverages and vitamin complexes.

Hereinafter, preferred embodiments of the present invention are described. However, the following examples are only preferred embodiments of the present invention, and the present invention is not limited to the following examples.

Example  1: Preparation of herbal composition

Example  1-1: Preparation of Active Extract

After washing well with tap water to remove impurities, 280g of the dried and dried throughout the day, chopped 2L of 70% (v / v) ethanol aqueous solution and 2 hours of reflux extraction apparatus [Heating mantle (manufacturer: GLASS-COL), Flask (manufactured by Corning), Condenser (manufactured by: Corning)] and filtered in Whatman No. 2 and concentrated under reduced pressure to obtain an ethanol extract. 250 ml of saturated ethyl acetate was added to the ethanol extract, and three layers were separated. Then, only ethyl acetate layer was collected and concentrated under reduced pressure to obtain a dried product at a temperature of 60 to 70 ° C. to obtain 12.75 g of strongly dried material.

Example  1-2: Preparation of Injin Extract

1.74 g of phosphorus dried product was obtained in the same manner as in Example 1-1 with respect to 85 g of phosphorus washed well with tap water to remove impurities and dried for one day.

Example  1-3: Leaflets  Preparation of Extract

2.45 g of foliar dried was obtained in the same manner as in Example 1-1 with respect to 85 g of foliar dried well in the shade.

Example  1-4: Preparation of Herbal Composition

The active extract, phosphorus extract and leaflet extract obtained in Examples 1-1 to 1-3 were mixed at a weight ratio of 5: 3: 2 to prepare 100 g, and then dissolved in 250 ml of saturated ethyl acetate solution, and then concentrated under reduced pressure. 50 ml of distilled water was added to the resultant obtained by concentration under reduced pressure, and then suspended and freeze-dried to obtain 3 g of the crude drug composition in powder form.

Example  2: Preparation of Herbal Composition

Prepare 50g of dried vigor, 30g of phosphorus, and 20g of leaflets (weight ratio 5: 3: 2), add 2.5L of 70% aqueous ethanol solution, and reflux extraction apparatus for 2 hours (Heating mantle [GLASS-COL], Flask [ Manufacturer: Corning], Condenser [manufacturer: Corning]), filtered through Whatman No. 2, and the filtrate was concentrated under reduced pressure to obtain 24.46 g of ethanol extract. 250 ml of saturated ethyl acetate solution was added to 24.46 g of the obtained ethanol extract, and the mixture was separated into three layers. Then, the ethyl acetate layer was collected and concentrated under reduced pressure until the herbal extract was dried at 60 to 70 ° C. Finally, 50 ml of distilled water was added, suspended, and freeze-dried to obtain 3.88 g of a crude drug composition in powder form.

Example  3: Preparation of herbal composition

Prepare 30g of dried vigor, 30g of phosphorus, and 30g of leaflet (weight ratio 1: 1: 1), and put it in 2.5ℓ of 70% ethanol aqueous solution and reflux extraction apparatus (Heating mantle [manufacturer: GLASS-COL], Flask [2 hours) Manufacturer: Corning], Condenser [manufacturer: Corning]), and filtered through Whatman No. 2, the filtrate was concentrated under reduced pressure to obtain 21.04 g of ethanol extract. 250 ml of saturated ethyl acetate solution was added to 21.04 g of the obtained ethanol extract, and the mixture was separated into three layers, and only ethyl acetate layer was collected and concentrated under reduced pressure until the herbal extract was dried at 60 to 70 ° C. Finally, 50 ml of distilled water was added, suspended, and freeze-dried to obtain 4.02 g of the crude drug composition in powder form.

Experimental Example  1: vigor, injin, Lobular  Of individual extracts and herbal compositions Anti-inflammatory  effect

Raw 264.7 cells, a mouse-derived macrophage line, were purchased from the Bank of Korea Cell Line and used for 10% fetal bovine serum (Fetal Bovine Serum, 26140-079. Manufactured by Gibco), 100 U / ml penicillin, and 10 µg / ml streptomycin. 2 x 10 ⁶ cells were inoculated into a 24 well plate containing DMEM medium containing and cultured at 37 ° C., 5% CO ₂ , and wet conditions for 24 hours.

In order to induce nitrogen monoxide in osteoarthritis in RAW 264.7 cells, E. coli lipopolysaccharide (LPS, Lipopolysaccharide), a cell wall component, was administered at a concentration of 1 ㎍ / ml, and the control group was treated with only DMSO as a solvent. The compositions of Examples 1-1, 1-2, 1-3, 1-4, 2, and 3 were each administered at a concentration of 20 μg / ml, followed by incubation for 16 hours. Then, the medium and the grease reagent were mixed at a ratio of 1: 1 and left at room temperature for 10 minutes, and then the absorbance at the wavelength of 540 nm was measured by using an absorbance meter.

Experimental Example  2: mouse In macrophage lines  Inhibitory effect of NO production

In order to confirm the anti-inflammatory action of the herbal compositions of Examples 1-4, 2 and 3, experiments were conducted to inhibit the production of the inflammatory reactant nitrogen monoxide in the macrophage lines of mice. Raw 264.7 cells, a mouse-derived macrophage line, were purchased from the Bank of Korea Cell Line and used for 10% fetal bovine serum (Fetal Bovine Serum, 26140-079. Manufactured by Gibco), 100 U / ml penicillin, and 10 µg / ml streptomycin. 2 x 10 ⁶ cells were inoculated into a 24 well plate containing DMEM (Dulbecco modified Eagle medium) medium and cultured at 37 ° C., 5% CO ₂ , and wet conditions for 24 hours.

Lipopolysaccharide (LPS), a cell wall component of Escherichia coli, was administered at a concentration of 1 ㎍ / ml to induce nitrogen monoxide in osteoarthritis in RAW 264.7 cells, and only DMSO (dimethyl sulfoxide) was used as a control in the control group. The experimental groups were treated with the compositions of Examples 1-4, 2, and 3 at concentrations of 0.8 μg / ml, 4 μg / ml, and 20 μg / ml, respectively, and then cultured for 16 hours. Then, the medium and the grease reagent were mixed at a ratio of 1: 1, and left for 10 minutes at room temperature, and then the absorbance was measured at an absorbance meter at a wavelength of 540 nm. The results are shown in FIGS. 2A to 2C.

2a to 2c, it can be seen that the herbal composition prepared according to the present invention inhibits the production of nitric oxide in a concentration-dependent manner in mouse macrophage Raw 264.7 cells. In addition, it can be seen that Examples 1-4 and Example 2 exhibited about 20% superior anti-inflammatory effect than that of Example 3.

Experimental Example  3: mouse In macrophage lines  Prostaglandin E ₂ (prostaglandin E ₂ , PGE ₂ ) Inhibitory effect on production

In order to confirm the anti-inflammatory action of the herbal compositions obtained in Examples 1-4, 2 and 3, the Cayman's PGE ₂ immunoassay kit (NO.514010) was used to investigate the inhibitory activity of the production of prostaglandin E ₂ , an inflammatory reactant in macrophages. It was carried out using.

In the same manner as in Experiment 2, mouse macrophages were cultured to administer LPS to a concentration of 1 µg / ml to 2 x 10 ⁶ cells, and the herbal compositions obtained in Examples 1-4, 2 and 3 were 0.8, 4, 20 Treated at a concentration of μg / ml and incubated for 16 hours. 50 μg of culture medium, 50 μl of PGE ₂ -AChE Tracer, and 50 μg of PGE ₂ Monoclonal Antibody were added to the enzyme-linked immunoassay kit and reacted at 4 ° C. for 18 hours. After the reaction, each well was washed and 200 μg of the color reagent Ellman s reagent was added to each well and reacted in the dark for 60 to 90 minutes. After the reaction, the absorbance was measured at a wavelength of 405 nm using an absorbance meter, and the results are shown in FIGS. 3A to 3C. In this case, LPS of FIG. 3 refers to the total amount of prostaglandin E ₂ generated after induction of osteoarthritis of the control group.

As shown in Figures 3a to 3c, the herbal composition of Example 1-4 inhibited PGE ₂ production in a concentration-dependent manner in mouse macrophages.

Experimental Example  4: Inhibitory effect of nitric oxide production in rabbit cartilage tissue

In order to confirm the anti-inflammatory action of the herbal compositions of Examples 1-4, 2 and 3, experiments were conducted to inhibit the production of inflammatory reactant nitrogen monoxide in cartilage tissue.

Tissue cells were separated from cartilage tissue from both hindlimb joints of 2-3 weeks old New Zealand white rabbits. 1 x 10 ⁶ rabbit cartilage in a 24 well plate containing DMEM medium containing 10% fetal bovine serum (26140-079. From Gibco), 100 U / ml penicillin, and 10 μg / ml streptomycin. Tissue cells were seeded and incubated under 37 ° C., 5% CO ₂ , wet conditions for 5-6 days. When the chondrocytes were grown to about 80%, the medium was changed to serum-free DMEM, and all the wells except the control were treated with Lipopolysaccharide-Cytokine Mixture (hereinafter LCM) for 20 minutes. Thereafter, only the control group and the negative control group treated only DMSO as a solvent, and the experimental groups of the herbal compositions of Examples 1-4, 2, and 3 were 0.8 μg / ml, 4 μg / ml, and 20 μg / ml, respectively. After administration, the cells were incubated for 16 hours. Then, the medium and the grease reagent were mixed at a ratio of 1: 1 and left at room temperature for 10 minutes, and then the absorbance was measured at an absorbance meter at a wavelength of 540 nm. The results are as shown in FIGS. 4A to 4C.

As shown in Figure 4a to 4c herbal compositions of Examples 1-4, 2 and 3 inhibited the production of nitric oxide in a concentration-dependent manner in rabbit cartilage tissue, which can be confirmed that the extract has an anti-inflammatory effect of osteoarthritis.

Experimental Example  5: prostaglandins in rabbit cartilage tissue E ₂  (prostaglandin E ₂ , PGE ₂ ) Inhibitory effect on production

In order to confirm the anti-inflammatory action of the herbal compositions of Examples 1-4, 2 and 3, Cayman's PGE ₂ immunoassay kit was used to perform the inhibitory activity of the production of the inflammatory reactant prostaglandin E ₂ in rabbit cartilage tissue.

In the same manner as in Experiment 4, 1 × 10 ⁶ rabbit cartilage tissue cells were treated with LCM in all wells except the control and reacted for 20 minutes, and then the herbal compositions prepared in Examples 1-4, 2, and 3 were 0.8, 4, 20 [mu] g / ml were treated and incubated for 16 hours. 50 μl of culture medium, 50 μl of PGE ₂ -AchE2 Tracer, and 50 μl of PGE ₂ Monoclonal Antibody were added to the enzyme-linked immunoassay kit and reacted at 4 ° C. for 18 hours. After the reaction, 200ul of color reagent Ellman s reagent was added to each well and reacted in the dark for 60 to 90 minutes. After the reaction, the absorbance at the wavelength of 405 nm using an absorbance meter is shown in Figs. 5a to 5c.

As shown in Figures 5a to 5c, extracts prepared according to Examples 1-4, 2 and 3 inhibited the production of prostaglandin E ₂ in a concentration-dependent manner in the chondrocytes of rabbits.

Experimental Example  6: in rabbit cartilage tissue cells Proteoglycan  Degradation inhibitory effect

In order to test the protective effect of the joint tissues of the herbal compositions of Examples 1-4, 2 and 3, a proteoglycan degradation inhibitory activity test was performed.

Culture of rabbit cartilage tissue cells, cartilage tissue was isolated from rabbit joints of 2-3 weeks old, as in Experiment 3. Tissue cells isolated were inoculated in 1 x 10 ⁶ cells using DMEM medium containing 10% Fetal Bovine Serum (FBS), 100 U / ml Penicillin, and 10 μg / ml Streptomycin in 24 wells. Cells were incubated at 37 ° C. under 5% CO ₂ wet conditions for 5-6 days after inoculation. When rabbit cartilage tissue cells were grown about 80%, changed to SFM (Serum Free Media), treated with LCM (Lipopolysaccharide-Cytokine Mixture) to all wells except the control and reacted for 20 minutes, Examples 1-4, 2 The herbal extracts according to 3 and 3 were administered at concentrations of 0.8, 4, and 20 µg / ml, and then cultured for 16 hours.

Through the above experimental method, the decomposition of proteoglycans, which is a component of rabbit cartilage tissue, was induced, and thus, glucose aminoglycan (GAG), which is a degradation product of proteoglycans, was added to 1-9, -dimethylmethylblue colorant (1-9, -Dimethylmethylene blue dye (DMB) was measured for absorbance at 525 nm, and then quantified by Chondroitin sulfate. The result is as shown in FIG.

As shown in FIGS. 6A to 6C, the herbal extracts prepared according to Examples 1-4, 2, and 3 inhibited the concentration of proteoglycans from chondrocytes and increased concentrations of proteoglycans in synovial fluid in a concentration-dependent manner in cartilage tissue cells of rabbits. .

Experimental Example  7: Degrading Enzyme Mattress in Rabbit Cartilage Joint Tissue Cells Metalloproteina Article-9 ( MMP -9) activity inhibitory effect

Example 1-4 In order to confirm the inhibitory effect of the articular degrading enzyme activity of the herbal composition, the activity test of MMP-9 was performed.

Cartilage tissue was isolated from rabbit joints of 2-3 weeks of age, as in Experiment 4, and DMEM medium containing 10% fetal bovine serum (FBS), 100 U / ml penicillin and 10 μg / ml streptomycin in 24 wells was used. 1 x 10 ⁶ cells were inoculated. Cells were incubated at 37 ° C. under 5% CO ₂ wet conditions for 5-6 days after inoculation. When rabbit cartilage tissue cells grew about 80%, the cells were changed to serum free DMEM, treated with LCM in all wells except the control group, and reacted for 20 minutes. Example 1-4: The herbal composition was 0.8, 4, 20 µg / ml. Incubated for 16 hours after administration at the concentration of.

Supernatants were obtained by centrifuge from cells incubated for 16 hours to remove cell debris. The supernatant sample and dye were mixed and incubated at 37 ° C. for one hour. SDS-PAGE was performed at 100 V for 3 hours using a 10% Zymogram gel (Novex EC61752). The gel Renaturing process was shaken at room temperature three times for 30 minutes, the gel was soaked in Developing buffer, and then developed in a 37 ° C. incubator for 48 hours. After the reaction, the gel was stained using Coomassie G blue dye, and the rest of the band except the band was decolorized, and the MMP-9 band of 92 kDda was confirmed and shown in FIG. 7.

As shown in FIG. 7, the herbal composition of Example 1-4 inhibited the activity of articular tissue degrading enzyme MMP-9 in a concentration-dependent manner.

Experimental Example  8: Inhibitory Effects of Acute Inflammatory Factors in Rabbit Cartilage Tissue Cells

Western blot to determine the effect of the herbal composition prepared in Examples 1-4 on the expression of cyclooxygenase 2 (hereinafter referred to as 'COX-2'), an enzyme that synthesizes prostaglandin, a pain-causing substance Western blot method was used.

Experimental conditions for rabbit cartilage tissue cells were prepared in the same manner as in Experiment 4. Example 1-4 Remove the culture medium of the cultured cells treated with the herbal composition and LCM and extract buffer (0.32M sucrose [S0389, manufactured by Sigma], 0.2M HEPES (N-2-hydroxyethylpiperazine-N-2) -dthanesulfonic acid) [H3375, manufactured by Sigma], 1 mM ethylenedia mintetraacetic acid (EDTA) [808288, manufactured by BM], 1 mM PMSF (phenylmethylsulfonyl fluoride) [P7627, manufactured by Sigma], 10 ug / ml Aprotinin [Aprotinin] [ A1153, manufactured by Sigma], 10ug / ml leupekin [L0649, manufactured by Sigma]), and the protein was quantified, and 20 ㎍ of the protein was subjected to SDS (sodium dodecyl sulfate) 10% polyacrylamide gel electrophoresis. Was carried out. The electrophoretic protein was transferred to PVDF (polyvinylidene difluoride, IPVH00010, manufactured by Millipore) membrane and reacted with 5% NFDM (non fat dry milk) solution, followed by primary and secondary antibody, followed by ECL. Color development with reagent (Amersham Biosciences) was exposed to X-ray film. 2 μg / ml of COX-2 (sc-1745, Santacruze) was used as the primary antibody, and Anti-Rabbit-IgG-HRP (sc-2004, Santacruze) 80 ng / ml was used as the secondary antibody. The results are shown in FIG. 8 using.

As shown in Figure 8, the herbal composition prepared according to Example 1-4 can be seen that the expression of COX-2 up to the level of steady state in the cartilage tissue cells of rabbits shows a very good effect.

Experimental Example  9: Acetogenic Mouse Weiding  Syndrome suppression effect

In order to confirm the analgesic inhibitory effect of the herbal compositions prepared in Examples 1-4 and 2, the symptoms of winding by the acetate of Whittle (1957) were observed in mice.

To the mice (ICR-based, Institute of Cancer Research) oral administration of the herbal composition prepared in Examples 1-4 and 2 per 100, 500, 1000 mg / kg mouse, 0.7% acetic acid-physiological saline solution after 30 minutes Intraperitoneal injection in an amount of 0.1 ml / 10 g. The number of guiding symptoms was observed from 10 minutes after injection to 10 minutes. The results are shown in Table 1. As a control, all experiments were performed in the same manner except that the herbal composition of the present invention was administered.

Group Dose (mg / kg) Average number of rides % Inhibition) Control - 20 - Example 1-4 200 15 25 Example 1-4 400 8.3 58.3 Example 2 200 15.7 22 Example 2 400 8 60

As shown in Table 1, the herbal compositions prepared according to Examples 1-4 and 2 can be seen that the analgesic effect is excellent as the number of guiding in the mouse is reduced by 20 to 60%.

Experimental Example  10: In the rat  Inhibitory effect on acute inflammation

In order to confirm the effect on the anti-inflammatory effect of the herbal composition prepared in Examples 1-4 and 2 using rat carrageenan (carrageenan) was confirmed the effect on the foot edema rate after inducing inflammation.

In the Spraque-Dawley rats fasted overnight, 200 ml of 400 mg / kg of the herbal composition was orally administered, and 1 hour carrageenan prepared by dissolving in physiological saline was injected subcutaneously in the right hind paw with 0.1 ml per foot. . Thereafter, the swelling degree of the right hind paw was measured at 1 hour intervals for 5 hours using a plethymometer. The degree of inhibition of edema is shown in FIG. 9. Rats of the control group were not orally administered the herbal composition.

As shown in Figure 9 herbal compositions prepared according to Examples 1-4 and 2 was excellent in the inhibitory effect by reducing the increase rate of foot edema.

Experimental Example  11: Test of the decomposition effect of harmful free radicals

SOD to protect the living body by removing the active oxygen generated by xanthine-xanthine oxidase to confirm the degradation effect on the harmful active oxygen of the herbal composition prepared in Examples 1-4 and 2 (Super Oxide Dismutase) was measured using a Cayman Superoxide Dismutase Assay Kit (Cat No. 706002) to compare the degree of decomposition of harmful active oxygen. As a comparative example (positive control), SK Pharmaceutical's tablet (trade name: Joins Tablet) was used, and the negative control was measured for the sample without adding the herbal composition.

200 ul of a noxious active oxygen detector (radical detector ) was added to each well, and 10 ul of the herbal composition prepared in Example 1-4 and 10 ul of SOD standard were added. Subsequently, xanthine-xanthine oxidase was added to generate noxious oxygen radicals, followed by incubation at room temperature for 20 minutes, and the respective absorbances were measured at 540 nm. Table 2 shows the degree of decomposition. At this time, Table 2 is a measured SOD value with 0.7 as a correction value measured in the negative control group. As shown in Table 2 below, the herbal compositions prepared according to Examples 1-4 and 2 can be seen that the decomposition of harmful active oxygen is excellent.

SOD (μg / ml) Example 1-4 Example 2 Comparative example 20 0.59 0.48 0.69 40 0.98 1.02 0.92 80 1.55 1.51 1.15 100 2.18 2.42 1.47

Experimental Example  12: formalin test

The formalin test was conducted to confirm the analgesic effect of the herbal extracts obtained in Examples 1-4 and Example 2. As a control, a test was performed on the mice that did not receive the herbal extract.

SC injection is performed in the left foot of the mouse's hind paw and placed in an acrylic observation chamber (20cm, high, 20cm, diameter) for 40 minutes and observed for behavior such as patting or licking the injected sole. Recorded at 10. 40 minutes is referred to as the first 5 min the 1 ^st phase, 1 ^st phase 2 ^nd phase the remaining 20 minutes from 15 minutes after of during.

As shown in Figure 10, the herbal extracts of Examples 1-4 and Example 2 can be seen that the analgesic effect is excellent.

Experimental Example  13: Observation of chondrocyte proliferation effect

In order to confirm the cartilage regeneration effect of the herbal composition prepared by Example 2, anterior cruciate ligament (ACLT) was performed on the joints of both hind limbs of the New Zealand white rabbit, and 5 × for 2 weeks after 3 days. After inducing arthritis by continuously exercising in a space of 5 m 3, Example 2 was orally administered for one month. After the experiment was completed, the joint cartilage of rabbits were collected and compared to the effect of cartilage cell proliferation. As a control, articular cartilage of rabbits not administered the herbal composition was collected and observed.

As shown in FIG. 11, in the rabbit orally administered to Example 2, the surface of the articular cartilage was smooth in appearance, whereas in the control group, the surface was very irregular and rough due to articular cartilage damage. From this, when the herbal extract of Example 2 is administered, it can be seen that the regeneration effect of damaged cartilage cells is excellent.

Experimental Example  14: Arthritis Induced Experimental Animals Joint synovial fluid  Gross measurement

The amount of synovial fluid has been reported according to the degree of osteoarthritis. After inducing osteoarthritis as in Experimental Example 13, the total amount of synovial fluid was administered at 2 and 4 weeks of oral administration to know the change in the amount of synovial fluid according to Example 2, respectively. Measured. As a control, after inducing osteoarthritis by the above method, the amount of the joint synovial fluid of the rabbit which was not administered the herbal composition was measured.

The amount of synovial fluid azo arsenic to a concentration of Ca ²⁺ in the synovial fluid Ⅲ ignition method (Arsenazo Ⅲ complexion method) [Micaylova Ⅴ. Et al., Anal. Chim. Acta, 53 (194), 1971], and the total amount of activity was calculated using the Donnan equilibrium equation. 1 ml of Arsenazo III reagent (manufactured by Sigma, Inc.) and 0.01 ml of the joint synovial fluid were mixed, left at room temperature for 5 minutes, and the absorbance was measured at 600 nm. The results of this experimental example are shown in Figure 12 and Table 3.

As shown in FIG. 12 and Table 3, the control group increased to 0.76 ml, but in rabbits administered orally to Example 2, a significant effect of inhibiting the volume increase of synovial fluid was reduced by increasing the width to 0.29 ml when compared to the control group. It can be seen that there is a.

0 weeks (ml) 2 weeks (ml) 4 weeks (ml) Normal 0.23 - - Control 0.58 0.39 0.76 Example 2 0.36 0.38 0.29

Experimental Example  15: Arthritis Induced Experimental Animals Joint synovial fluid  Determination of Proteoglycan

In the case of administering the herbal composition of Example 2 obtained in Experimental Example 14 and using the joint synovial fluid of the control group, the proteoglycan concentration in the synovial fluid was measured in the same manner as in Experimental Example 6 to determine the effect of protecting the joint tissue. The results measured in this experimental example are shown in Figure 13 and Table 4.

As shown in FIG. 13 and Table 4, the control group increased by 60.12% to 16.38 μg / μl, but in the group administered orally for 4 weeks, the increased proteoglycan content in the synovial fluid decreased by about 35.98% to 7.33 μg / μl. It can be seen that it has a good effect on the protection of joint tissue.

Week 0 2 weeks 4 Weeks Normal 8.20 - - Control 10.23 11.96 16.38 Example 2 11.45 10.65 7.33

Experimental Example  16: Arthritis Induced Experimental Animals Joint synovial fluid Total protein (Total Protein) Volume Measurement

As osteoarthritis progressed, cartilage ruptured and total protein was released into the synovial fluid. Therefore, total protein concentration in the synovial fluid was measured to investigate the inhibition of osteoarthritis. In the case of administering the herbal composition of Example 2 obtained in Experimental Example 14 and the control of the joint synovial fluid, the total protein concentration in the synovial fluid was measured at 595nm by the Bradford method (absorbance), in this Experimental Example The measured result was shown in FIG. 14 and Table 5.

As shown in FIG. 14 and Table 5, the total protein concentration in the synovial fluid increased by 24.99% to 52.71 μg / μl in the normal range of 25.67 μg / μl in the control group, but 38.62 in the oral administration of Example 2 for 4 weeks. 26.68% of inhibition was performed at μg / μl, indicating a great effect.

Week 0 2 weeks 4 Weeks Normal 25.67 - - Control 42.17 46.96 52.71 Example 2 52.67 41.68 38.62

Experimental Example  17: Arthritis Induced in Experimental Animals Joint synovial fluid  Prostaglandin E ₂ (Prostaglandin E ₂ , PGE ₂ Volume measurement

In the case of administering the herbal composition of Example 2 obtained in Experimental Example 14 and using the joint synovial fluid of the control group, the concentration of prostaglandin E _{2 in} the synovial fluid was measured in the same manner as in Experimental Example 5 to determine the anti-inflammatory effect in the joint tissue. . The results measured in this experimental example are shown in Figure 15 and Table 6.

As shown in FIG. 15 and Table 6, the concentration of prostaglandin E ₂ in synovial fluid increased by 221.78% from 0.65 μg / μl to 2.1 μg / μl in the control group, but increased by 25.21% in the group administered orally to Example 2 for 4 weeks. It showed a very large inhibitory effect compared to the control group.

Week 0 2 weeks 4 Weeks Normal 0.33 - - Control 0.65 0.96 2.10 Example 2 0.63 0.44 0.79

Experimental Example  18: joint tissue Safranin -O ( Safranin -O) staining results and measurement of synovial cell distribution

After inducing osteoarthritis in the same manner as in Experimental Example 13, the herbal composition of Example 2 was orally administered at a concentration of 200 mg / kg for one month. After that, the distal femur of the rabbit was collected and fixed in 4% formalin (Sigma, F0507) for more than 24 hours, and the surface to be sliced was cut into 5 mm thickness and dehydrated with 5% nitric acid for 24 hours and then made into a paraffin block. The distribution and surface of the synovial membrane cells were examined through histochemistry analysis, in which 4 μm flakes were stained with safranin-O (Sigma S2255), a specific stain of cartilage tissue. The results measured in the present experimental example are shown in FIG. 16. As a control, the distal femoral synovial membrane cells of rabbits that did not receive the herbal composition were measured.

As shown in FIG. 16, in the group administered orally for 4 weeks to Example 2, when the osteoarthritis stained in red compared to the control group, blue-stained sites indicating thick cells were observed on the cells. It can be seen that the herbal composition has an effect of cartilage regeneration.

Formulation example  1: preparation of tablets

100 mg of the herbal composition prepared according to Example 2, 90 mg of corn starch, 180 mg of lactose, 18 mg of L-hydroxypropyl cellulose, 5 mg of polyvinylpyrrolidone and a suitable amount of ethanol were granulated by wet granulation. Then, 1.8 mg of magnesium stearate was added and mixed, and the tablets were compressed to 400 mg.

Formulation example  2: Preparation of Soft Capsule

100 mg of herbal composition prepared according to Example 2, 180 mg of soybean oil, 40 mg of sulfur oil, 128 mg of coconut oil, 20.5 mg of soybean phospholipid, 212 mg of gelatin, 50 mg of glycerin (specific gravity 1.24), 76 mg of d-sorbitol, 0.54 mg of paraoxybenzoic acid methyl, 0.90 mg of paraoxybenzoic acid propyl, 0.56 mg of methylvanillin and yellow 203 were prepared to be contained in one capsule according to the method of soft capsule in the Pharmacopeia General Formulation.

Formulation example  3: Manufacture of capsule

100 mg of the herbal composition prepared according to Example 2, 83 mg of corn starch, 175 mg of lactose and 2 mg of magnesium stearate were homogeneously mixed and filled to contain 360 mg in one capsule.

Formulation example  4: manufacturing of healthy foods and beverages

A health food or beverage composition containing the herbal composition prepared according to Example 2 as an active ingredient was prepared as follows.

Formulation example  4-1 Manufacturing of health food

Example 2 The herbal composition prepared according to 1000 mg, vitamin A acetate 70 ug, Vitamin E 1 mg, vitamin B ₁ 0.13 mg, Vitamin B ₂ 0.15 mg, Vitamin B ₆ 0.5 mg, Vitamin B ₁₂ 0.2 ug, Vitamin C 10 mg, biotin 10 ug, nicotinamide amide 1.7 mg, folic acid 50 ug, calcium pantothenate 0.5 mg, ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, potassium monophosphate 15 mg, potassium diphosphate 55 mg Health food was prepared by the conventional method with a composition and content of 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride.

The composition ratio of the above-mentioned vitamin and mineral mixtures is a composition suitable for a relatively healthy food in a preferred embodiment, but the composition ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for producing a health food composition according to the method of general purpose.

Formulation example  4-2 Preparation of Healthy Drinks

100 mg of the herbal composition prepared according to Example 2, 15 g of vitamin C, 100 g of vitamin E (powder), 19.75 g of iron lactate, 3.5 g of nicotinamide, 3.5 g of zinc oxide, 0.2 g of vitamin A, 0.25 g of vitamin B1, A health beverage was prepared using conventional methods with 0.3 g of vitamin B2 and water quantitative as the composition and content.

After mixing the above components in accordance with a conventional method for preparing healthy beverages, and stirring and heating at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and refrigerated Used to prepare a healthy beverage composition of the invention.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

Formulation example  4-3: Chewing gum  Produce

Chewing gum was prepared using a conventional method using 0.24 to 0.64% of the herbal composition prepared according to Example 2, 20% of gum base, 76.36 to 76.76% of sugar, 1% of fruit flavor, and 2% of water.

Formulation example  4-4: Preparation of Ice Cream

Herbal medicine composition prepared according to Example 2 0.24 ~ 0.64%, milk fat 10.0%, nonfat milk solids 10.8%, sugar 12.0%, starch syrup 3.0%, emulsifying stabilizer (span, span) 0.5%, fragrance (strawberry) 0.15% and Ice cream was prepared using a conventional method with a composition and content of 62.91-63.31% of water.

Formulation example  4-5: Preparation of Beverages

The herbal composition prepared according to Example 2 0.48-1.28 mg, honey 522 mg, thioctoamide amide 5 mg, nicotinic acid amide 10 mg, riboflavin sodium 3 mg, pyridoxine hydrochloride 2 mg, inositol 30 mg, ortho acid 50 mg and A beverage was prepared using conventional methods with a composition and content of 200 ml of water.

The composition containing the active ingredient of the active, phosphorus and leaflet of the present invention as an active ingredient is anti-inflammatory action, anti-inflammatory analgesic action, inhibiting articular tissue degrading enzyme, inflammation-related enzyme inhibitory action, inhibiting foot edema, inhibiting the increase of joint synovial fluid It can be used as a preventive or therapeutic agent for cartilage regeneration and osteoarthritis due to its therapeutic effect such as inhibiting the increase of total protein of joint synovial fluid. In addition, it can be used as a pharmaceutical or food composition containing the active ingredient.

Claims (10)

The composition for cartilage regeneration comprising ethanol, distilled water or extract of these mixtures, ethanol, distilled water or extracts of these mixtures, and extracts of ethanol, distilled water or mixtures of these leaves as active ingredients. The composition of claim 1, wherein the composition comprises 5 to 50 parts by weight of active ethanol, distilled water or extracts of these mixtures, 3 to 30 parts by weight of extracts of ethanol, distilled water or mixtures of these phosphorus and ethanol of the leaflets. Cartilage regeneration composition comprising 2 to 20 parts by weight of distilled water, or extracts of these mixtures. The composition for cartilage regeneration according to claim 1, wherein the composition for cartilage regeneration is extracted with distilled water or ethanol aqueous solution, or a mixture of vigor, phosphorus and leaflets, and then fractionally extracted with ethyl acetate. The composition of claim 1, wherein the composition for cartilage regeneration is useful for cartilage regeneration, characterized in that to inhibit the degradation of proteoglycans. The composition for cartilage regeneration according to claim 1, wherein the composition for cartilage regeneration is selected from the group consisting of oral tablets, oral soft capsules, ointments, injections or transdermal administrations. 5 to 50 parts by weight of the extract of ethanol, distilled water, or a mixture of these, 3 to 30 parts by weight of the extract of ethanol, distilled water, or a mixture thereof, and 2 to 20 parts by weight of the extract of ethanol, distilled water, or a mixture of these leaves. Osteoarthritis treatment composition comprising as an active ingredient. The composition for treating osteoarthritis of claim 6, wherein the composition for treating osteoarthritis is extracted with distilled water or an aqueous ethanol solution or a mixture of vigor, phosphorus and leaflets, and then fractionated with ethyl acetate. 7. The composition for treating osteoarthritis of claim 6, wherein the composition for treating osteoarthritis reduces the amount of increased proteoglycan, prostaglandin, matrix metalloprotease-9 (MMP-9), and cyclooxygenase 2 in the blood, which are markers of osteoarthritis development. Osteoarthritis treatment composition. According to claim 6, The osteoarthritis therapeutic composition is a composition for treating osteoarthritis selected from the group consisting of oral tablets, oral soft capsules, ointments, injections and transdermal administration. Extracting by extracting one or more solvents selected from the group consisting of distilled water, alcohol having 1 to 5 carbon atoms, and ethyl acetate, respectively or by mixing the active, phosphorus and leaflets; And Method of producing a composition for treating cartilage regeneration and osteoarthritis comprising the step of concentrating the extract with ethyl acetate layered.

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