WO2007007958A1

WO2007007958A1 - Herbal pharmaceutical composition for regenerative agent of cartilaginous tissue and treatment of osteoarthritis

Info

Publication number: WO2007007958A1
Application number: PCT/KR2006/002439
Authority: WO
Inventors: Sung-Yeoun Hwang; Si-Na Kim; Gyeong-Sug Nam; Sung-Wan Hwang
Original assignee: Yuhan Corporation
Priority date: 2005-07-08
Filing date: 2006-06-23
Publication date: 2007-01-18

Abstract

The present invention relates to a herbal pharmaceutical composition for regenerating cartilaginous tissue and treating osteoarthritis. More specifically, the invention relates to the composition comprising an Angelica koreanum extract, an Artemisia apillaries extract, and a Perilla frutescens extract. The composition of the present invention has an efficacy in anti-inflammatory activity, anti-inflammatory and analgesic activities, and inhibitory activities on articular tissue protease, inflammatory enzymes, foot edema, increased synovial fluid in joints, and increase of whole proteins in articular synovial fluid, and the like, and thus can be used as an agent of regenerating cartilaginous tissue and an agent for treating osteoarthritis.

Description

TITLE OF THE INVENTION

HERBAL PHARMACEUTICAL COMPOSITION FOR REGENERATIVE AGENT OF CARTILAGINOUS TISSUE AND TREATMENT OF OSTEOARTHRITIS

BACKGROUND OF THE INVENTION (a) Field of the Invention

The present invention relates to a herbal pharmaceutical composition for regenerating cartilaginous tissue and treating osteoarthritis. More specifically, the composition has an efficacy in anti-inflammatory activity, anti-inflammatory and analgesic activities, and inhibitory activities on articular tissue protease, inflammatory enzymes, foot edema, increase of articular synovial fluid, and increase of whole proteins of articular synovial fluid, and the like, and thus can be used as an agent of regenerating cartilaginous tissue and an agent of treating osteoarthritis.

(b) Description of the Related Art

Although an aerobic organism including humans, has a self-defense system to reactive oxygen which is produced in energy metabolism using oxygen, the overproduction of the oxygen free radical over the self-defending capacity can cause various diseases such as arthritis. The reactive oxygen is a free radical produced by the reduction of triplet oxygen in ground state (³O₂), and includes superoxide anion (¹O₂-) of singlet oxygen, hyperoxide (H₂O₂), and hydroxyl radical ( OH). The reactive oxygen damages protein, DNA, enzyme and immune-related factors such as T cells, thereby causing various diseases. As a result, many anti-oxidants have been actively developed . There are many antioxidants including antioxidant enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase; natural antioxidants such as Vitamin E, Vitamin C₁ carotenoid, glutathione; synthetic antioxidants such as t-Butyl-4-hydroxyanisole (BHA), and 3,5-(t-Butyl)-4-hydroxytoluene(BHT). However, as the defending activity of natural antioxidant enzymes decrease with age, the synthetic antioxidant causes mutation or deveolps toxicity. Thus, the development of stable and effective antioxidants are still required.

An inflammatory disease is referred to as an abscessed disorder caused by bacterial infection. Two cyclooxygenases (COX) are known as an inflammatory enzyme. Cyclooxygenase-1 (COX-1 ) involves the production of prostaglandin (PG) in various normal organs and tissues, such as in the gastrointestinal tract and kidney, as well as in the inflammatory region. Cyclooxygenase-2 (COX-2) only acts in the inflammatory region. Non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac, aspirin, and ibuprofen which are commercially available inhibit both of COX-1 and COX-2, or inhibit COX-1 principally. As the continuous administration of NSAIDs inhibit PG which is essential for maintaining the functions of the liver, gastrointestinal tract, kidney, as well as COX, the NSAIDs cause many adverse side-effects (Isselbcher et al., Harrison's Principles of Internal Medicine, (13th ed)2, pp1543-1711).

Osteoarthritis (OA) is the most common disease and accounts for 40 to 60% of skeletal muscle system disease. As the average span of a man's life increases, a prevalence of osteoarthritis increases continuously. At present, about 15% of the world's population suffers from osteoarthritis, and 60% of patients are in their sixties (Haq I et al., J.Osteroarthritis, 79(993), pp377~383).

Osteoarthritis is related to the wearing-out of articular cartilage, which leads to direct bone-to-bone contact and causes joint pain and prevents body activity. As the chronic disease needs long-term care, long-term drug administration develops complications and side-effects. Accordingly, a drug is still required for treating such complications as well as for pain reduction, and the maintenance and recovery of articular cartilage function in the treatment of arthritis. In general, a method of treating arthritis is brought by focusing on alleviating the pain by administering a simple pain-killing drug at an early state where only pain is present. In the subsequent stages where the pain is felt continuously due to the process of the disease, anti-inflammatory agents with analgesic activity such as corticosteroids have been used. For example, anti-inflammatory agents such as hydrocortisone and betamethasone are administered over a long time, but they weaken the functions of the liver, stomach, and kidney, and inhibit the generative ability of cartilage cells. Thus, under continuous observation, the anti-inflammatory agents must be administered by injecting into the joint, and not by oral administration.

There are many drugs which lower the side-effects and have anti-inflammatory and analgesic activity for treating arthritis. Representative examples are NSAIDs such as diclofenac, aspirin and ibuprofen. NSAIDs cause side effects in digestive organs, especially the stomach and intestines, and only remove the pain and inflammation without treating arthritis completely.

SUMMARY OF THE INVENTION

To resolve the problems in the prior arts, an objective of the present invention is to provide a composition for regenerating the cartilaginous tissue and treating osteoarthritis with efficacy in anti-inflammatory activity, anti-inflammatory and analgesic activities, and inhibitory activities on articular tissue protease, inflammatory enzymes, foot edema, increase of articular synovial fluid, and increase of whole proteins of synovial fluid, and the like.

Another objective of the present invention is to provide a composition for regenerating the cartilaginous tissue and treating osteoarthritis comprising an Angelica koreanum extract, an Artemisia apillaries extract, and a Perilla frutescens extract as an effective agent.

The further objective of the present invention is to provide a method of preparing a composition for regenerating cartilaginous tissue or a composition for treating osteoarthritis comprising the steps of extracting a plant mixture or each plant of Angelica Korean, Artemisia capillaries, and the Perilla frutescens, with at least a solvent selected from the group consisting of distilled water, C1 to C5 alcohol, ethyl acetate and a mixture thereof; and concentrating the extracts by performing a fractional extraction and separating the fraction of ethyl acetate.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows an anti-inflammatory effect of each extract of Angelica koreanum, Artemisia apillaries, and Perilla frutescens, and a herbal composition comprising their mixture on the osteoarthritis caused by lipopolysacharide (LPS) in accordance with Example 1-1 to 1-3, Example 2, and Example 3.

Figs. 2A to 2C represent the carbon monoxide (CO) production after treating mouse macrophage having osteoarthritis caused by LPS with the herbal compositions obtained in Example 1-4, Example 2 and Example 3 in an amount of

0.8, 4, 20μg/mi, in accordance with Experimental Example 2, and the LPC indicates the CO production in a control group.

Figs. 3A to 3B represent the prostaglandin E₂ production after treating mouse macrophage having osteoarthritis caused by lipopolysacharide-cytokine-mixture (LCM) with the herbal compositions obtained in

Example 1-4, Example 2, and Example 3 in an amount of 0.8, 4, and 20 μg/mf,, respectively in accordance with Experimental Example 3, and CM indicates the prostaglandin E₂ production in a control group.

Figs. 4A to 4C represent the nitrogen monoxide (NO) production after treating rabbit cartilaginous tissue having osteoarthritis caused by LCM with the herbal compositions obtained in Example 1-4, Example 2, and Example 3 in an amount of 0.8, 4, 2OjUgM , and CM in FIG. 4A and 4B indicates the CO production in a control group.

Figs. 5A to 5C represent the prostaglandin E₂ production after treating rabbit cartilaginous tissue having osteoarthritis caused by LCM with the herbal compositions obtained in Example 1-4, Example 2, and Example 3 in an amount of

0.8, 4, IQμglxd, in accordance with Experimental Example 5, and the CM in the FIGs. indicate the prostaglandin E₂ production after causing osteoarthritis in a control group by using LCM. Figs. 6A to 6C represent the decomposed proteoglycan after treating cartilaginous tissue having osteoarthritis caused by LCM in accordance with Experimental Example 6 with the herbal composition obtained in Example 1-4,

Example 2 ,and Example 3 in an amount of 0.8, 4, 2O#g/m<5, and the CM in the FIGs. indicate the produced proteoglycan after causing osteoarthritis in a control group by using LCM.

Fig. 7 represents the total amount of activated MMP-9 and MMP-2 after treating cartilaginous tissue having osteoarthritis caused by LCM with the herbal compositions obtained in Example 1-4, Example 2, and Example 3 in an amount of 0.8, 4, 20μg/m£, in accordance with Experimental Example 7, and the CM indicates the activated MMP-9 after causing osteoarthritis in a control group by using LCM.

Fig. 8 represents the total amount of expressed COX-2 after treating rabbit cartilaginous tissue having osteoarthritis caused by LCM with the herbal composition obtained in Example 1-4, Example 2, and Example 3 in an amount of 0.8, 4, 20μg/mϋ in accordance with Experimental Example 8, and CM indicates the total amount of expressed COX-2 after causing osteoarthritis in a control group by using LCM.

Fig. 9 shows an inhibitory effect of herbal compositions obtained in Example 1-4 and Example 2 on rat food edema in accordance with Experimental Example 9

Fig. 10 shows an inhibitory effect of herbal compositions obtained in Example 1-4 and Example 2 on pain in accordance with Experimental Example 10.

Fig. 11 shows an effect of herbal composition obtained in Example 2 on a regeneration of cartilaginous tissue of an osteoarthritis-derived animal.

Fig. 12 shows an effect of herbal composition obtained in Example 2 on the change in amount of the articular synovial fluid of an osteoarthritis-derived animal. Fig. 13 shows an effect of herbal composition obtained in Example 2 on proteoglycan in the articular synovial fluid of an osteoarthritis-derived animal.

Fig. 14 shows an effect of herbal composition obtained in Example 2 on whole proteins in the articular synovial fluid of an osteoarthritis-derived animal.

Fig. 15 shows an effect of herbal composition obtained in Example 2 on proteoglycan E2 in the articular synovial fluid of an osteoarthritis-derived animal.

Fig. 16 shows a result of an Safranin-0 staining analysis and cell distribution of the synovial fluid membrane in the articular tissue of an osteoarthritis-derived animal which were treated with the herbal composition obtained in Example 2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in more detail as below. In the present invention, the composition for regenerating the cartilaginous tissue and treating osteoarthritis includes an extract of Angelica koreanum, an extract of Artemisia apillaries, and an extract of Perilla frutescens as an effective agent, and more preferably, an extract of Angelica koreanum which is prepared by extracting with ethanol, distilled water, or a mixture thereof, an extract of Artemisia apillaries which is prepared by extracting with ethanol, distilled water, or a mixture thereof, and an extract of Perilla frutescens by extracting with ethanol, distilled water, or a mixture thereof. Ostericum koreanum max. used as an active agent in the present invention contains oil components such as limonene, o-cresol, and alpha-pinene, osthenol, and bergaptin, and thus has efficacy for colds, headaches, neuralgia, and the like.

Artemisiae Capillaris Thunb. is a perennial plant belonging to Chrysanthemum frutescens, and is also called Artemisa sacrorum subsp. Kitamura, Artemisa sacrorum subsp. manshuria var. vestita Kitamura, and the like. Due to its unique flavor and efficacy, Artemisiae Capillaris thunb. was first noticed. In general, it is used widely as a food, drug, and a single-medicine prescription. As described in Donguibogam (an old Korean oriental medicine book) and Bonchogangmok (an old Chinese botanical list), Artemisiae Capillaris has clinical efficacy in the recovery of liver damage; prevention and treatment of liver diseases such as fatty liver, liver cancer, hepatitis, and jaundice; treatment of gastrointestinal diseases such as aperitive, apepsia, cholecystitis, and gastroenteropathy; enhanced and circulatory diseases such as apoplexy, blood circulation and blood purification; treatment of diabetes. The main components in Artemisiae Capillaris are scopoletin of glucoside, oil component, coumarin, oxycarbonic acid, saponin, tannin, sitosterin resin, and the like, and thus Artemisiae Capillaris is expected to have detoxification, antibacterial activity, immunogenic activity, and etc. It reduces the blood lipid, and thus is used for the prevention and treatment of hypertension, diabetes, obesity, and brain strokes.

Perilla herba used in the present invention is Perilla frutescens, an annual plant belonging to Labiatae. In oriental medicine, the leaf and seeds are generally used. Oil of Perilla herba contains 55% of peril aldehyde, 20-30% of l-limonene and a small amount of α- pinene, arginine, cumic acid, and cyanidin-3-(6-p-cumaroyl-β-D-glucoside)-5-β-D-glucoside. Purified oil of perilla leaf includes isoegoma ketone. Perilla frutescens is used as a sweating agent, antitussive agent, stomachic agent, diuresis, calmative and analgesic agent. The aged stem of the perilla herba is located in the root or in the vicinity of the root and has been known to remove gout, sweating, and expectorant, and to treat dizziness, pain, congestion, and coughs.

The composition of the present invention includes 5 to 50 parts by weight of the extract of Ostericum koreanum, 3 to 30 parts by weight of the extract of Artemisiae Capillaris, and 2 to 20 parts by weight of the extract of Perilla frutescens, preferably. The extract of Ostericum koreanum, the extract of Artemisiae Capillaris, and the extract of Perilla frutescens can be contained in a weight ratio of 1 to 10: 1 to 6: 1 to 4 in the composition. The mixed ratios of the extracts are determined to consider desirable efficacy, side effects and the manufacturing costs. If the amount is outside of the range, the herbal composition is cytotoxic, and cannot provide the desired effect. The composition of the present invention has an efficacy in anti-inflammatory activity, anti-inflammatory and analgesic activities, and inhibitory activities on articular tissue protease, inflammatory enzymes, food edema, increase of articular synovial fluid, and increase of whole proteins of synovial fluid, and the like, and thus can be used as an agent of regenerating cartilaginous tissue and an agent of treating osteoarthritis. Examples of inflammatory diseases include osteoarthritis, rheum arthritis, omarthritis, tendinitis, tenosynovitis, myositis, and the like. The composition is very efficacious against osteoarthritis.

The parts of a plant to be extracted of Ostericum koreanum, Artemisiae Capillaris and Perilla frutescens can be the flower, leaf, stem, sheaths, flower bud, root, fruit, seed, and plant body, but preferably, the root of Ostericum koreanum, the stem and leaf of Artemisiae Capillaris, and leaf of Perilla frutescens.

The extracts of Ostericum koreanum, Artemisiae Capillaris and Perilla frutescens are prepared by pulverizing each plant or dried plant, extracting them with the addition of a solvent in a weight ratio of 1 : 5 to 1 : 10 under the sonication or reflux, and filtering or centrifuging to obtain supernatant. The solvent is at least one selected from the group consisting of water, C1 to C5 alcohol, ethyl acetate, and hexane, and preferably water, ethanol and a mixture thereof. The sonication is performed for 2 to 5 hours.

Preferably, the composition is prepared by extracting each plant of Ostericum koreanum, Artemisiae Capillaris and Perilla frutescens, or their plant mixture with distilled water, or aqueous ethanol, and filtering or centrifuging to obtain supernatant. More preferably, the preparation method further includes a step of fractional extraction by using ethyl acetate to produce ethyl acetate fraction. The fractional extraction can be done at least three times. The herbal composition can be used in liquid, vacuum concentrated, or powder obtained by freeze-drying.

The herbal composition can be provided in a pharmaceutically effective dosage form. The suitable dosage forms are powder, liquid, peel, tablets, sugar coated tablets, hard or soft capsules, solution, dispersion, emulsion, injections, suppository, and etc., but are not limited thereto. The suitable dosage form of composition can be prepared by using a pharmaceutically acceptable salt and pharmaceutically inactive organic or inorganic carrier. When the preparation is in a form of a tablet, coated tablet, sugar coated tablet, and hard capsule, it includes lactose, corn starch or its derivatives, talc, or stearic acid or its salt. In case of a soft tablet, the preparations can include plant oil, wax, lipid, semisolid and liquid polyol. In case of a solution or syrup, water, polyol, glycerol and plant oil can be used. The suppository carrier includes natural oil, hardened oil, wax, lipid, liquid polyol, and the like.

Optionally, the composition of the present invention further contains a preservative agent, stabilizer, wetting agent, emulsifier, solvent, sweetening agent, colorant, osmotic stabilizer, antioxidant, and the like. The method of administering the preparations can be selected suitably depending on the dosage form, for example, by oral or parenteral administration.

In addition, the herbal composition can be used as a food composition for the prevention or treatment of inflammatory diseases, or as a functional food for regenerating cartilaginous tissue. The composition can be any food which is commercially available, but is not limited thereto.

As an inflammatory reaction, which occurs in the process of cartilage destruction leading osteoarthritis, produces inflammation-related enzymes, it accelerates cartilage damage. In general, overproduction of nitric monoxide (NO) occurs in the inflammatory region, thereby accelerating cell necrosis [Moncada, S et al., Pharmacol. Rev. 43: 109-142 (1991 )]. It is important to inhibit the activity of inducible nitric oxide synthetase (iNOS) or its expression during the treatment of inflammatory diseases. iNOS produces NO excessively in a short time to defend against external stimulation. However, excessive production of NO in arthritis causes secondary adverse reactions such as cell necrosis or pain. Thus, the inhibition of iNOS over-expression is a key step in the treatment of osteoarthritis.

When osteoarthritis occurs, pain and an inflammatory response in the joints increase. The pain is caused by an increased concentration of prostagladin E₂ in the synovial fluid. To treat osteoarthritis, the concentration of prostagladin E₂ must be reduced in the synovial fluid.

Proteoglycan is a complex molecule including protein and sugar, and forms a condense structure with collageneous fibers in cartilage to bend and stretch the body. In addition, proteoglycan keeps moisture in cartilaginous tissue like a sponge, thus making the cartilage move continuously. In the process of osteoarthritis, the cartilage destruction and inflammatory reaction release proteoglycan into the synovial fluid, thereby increasing the amount of synovial fluid and proteoglycan concentration in the synovial fluid. Thus, the reduced concentration of proteoglycan in the synovial fluid is an important marker for measuring the improvement of osteoarthritis. If the release of proteoglycan into the synovial fluid is inhibited, proteoglycan is produced in cartilage, thereby affecting cartilage cell growth. The regenerative effects of cartilaginous tissue can be measured by the inhibition of proteoglycan release into the synovial fluid (Ailson M. Badger et al., Inhibition of lnterleukin-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 exampleal Therapeutics. Vol.290, 587-593). The cartilaginous tissue in osteoarthritis is destroyed by the production and activation of MMP, where the expression and activities of MMP-1 (collagenase-1 ), MMP-2, MMP-3 (stromelysin-1 ), MMP-8 (neutrophilcollagenase), MMP-9 (gelatinase), and MMP-13(collagenase-3), etc increase. The reduced concentration of MMP9 in synovial fluid is also a maker for measuring the improvement of osteoarthritis.

Enzyme COX-2 is an enzyme synthesizing prostagladin E₂ which causes pain. As COX-2 synthesis is promoted by other stimuli including NO, the inhibition of activity and expression of COX-2 is important for treating osteoarthritis. The reactive oxygen species that causes inflammation can be removed by an antioxidant enzyme of SOD, and thus SOD is a marker for measuring the improvement of osteoarthritis.

In aspects of the essential factors for the prevention or treatment of osteoarthritis as described above, the herbal composition of the present invention reduces the concentration of proteoglycan and prostagladin E₂ in the cartilaginous tissue by inhibiting their separation as this inhibits CO production and the expression of MMP-9 and COX-2, reduces writhing caused by acetic acid and food edema, and increases SOD to remove reactive oxygen species.

The herbal composition decreases the amount of synovial fluid, the total amount of proteins released into synovial fluid during the destruction of cartilage, and the concentration of proteoglycan in synovial fluid, thereby preventing the process of osteoarthritis.

The herbal composition can treat osteoarthritis completely by regenerating the damaged cartilage cell. The regenerated cartilage cell can be observed by appearance and Safranin-0 staining to show cell distribution and surface of the synovial fluid membrane. In general, the smooth surface of cartilaginous tissue is observed to determine whether the cartilage cell regenerates in cartilaginous tissue of osteoarthritis. In Safranin-O staining, the cartilaginous cell cannot be observed in osteoarthritis, but the cartilage cell growth and alleviation of osteoarthritis can be determined by observing cartilage tissue formation over the stained region and the staining degree shown in red compared to that in the control group. The composition can be administered orally or parenterally. The solid preparation for oral administration includes tablets, peels, powder, granules, capsules, and the like. The solid preparation contains at least an expedient, for example, starch, calcium carbonate, sucrose, lactose and gelatin, in addition to the herbal composition. The preparation further includes a lubricant such as magnesium stearate, or talc. The examples of liquid preparation for oral administration are dispersion, liquid, emulsion, syrup, and the like. The liquid preparation contains water, liquid paraffin, and other various diluents as a simple diluent, and a further wetting agent, sweetening agent, flavoring agent and preservative. The preparation for parenteral administration contains sterilized aqueous solution, propylene glycol, polyethylene glycol, plant oil such as olive oil, injectable ester such as ethyl oleate as an non-aqueous solvent and dispersing agent. The dosage unit, for example, contains 1/2, 1/3, 1/4, one, two, three or four times more than that of a dosage amount. An amount of dosage unit means the amount of active drug administered at a time, and usually all, 1/2, 1/3, or 1/4 of the amount administered for 1 day.

When the composition is used as a pharmaceutical composition, the dosage of an active agent is 1 to 1 ,000 mg/kg, and preferably 50 to 300 mg/kg, and can be administered 1 or 4 times for 1 day. The dosage can be determined in consideration of body weight, age, sex, health condition of the subject in need, diet, the time and method of administration, excretion rate, disease severity and the like.

In addition, the present invention relates to a food composition or a food additive comprising the extract of Angelica koreanum, the extract of Artemisia apillaries, and the extract of Perilla frutescens as an effective agent.

In case of the food composition, the extracts are used alone or in combination with other food additives according to the conventional method used in the prior art. The effective agents are contained in an amount of 1 to 99.9 weight %, and preferably 10 to 70 weight % of total food composition. The amount of effective agents can be selected in consideration with its use (e.g., prevention, therapy, or health). In order to use the composition for maintaining and controlling health over a long time, the amount can be lower than the range. This is because the effective agent does not have stability problem, and thus, can be contained more than the range.

In the present invention, there is no limit to the food that can be used. Examples of such foods are meat, sausages, bread, chocolate, candy, snack, sweet snacks, pizza, instant noodles, other noodles, gum, dairy foods such as ice cream, seasonings, soup, tea, drinks, alcohol beverage, and vitamin composites.

The present invention is further explained in more detail with reference to the following examples. These examples, however, should not be interpreted as limiting the scope of the present invention in any manner.

Example 1: Preparation of herbal composition

Example 1-1 : extract of Angelica koreanum

28Og of Angelica koreanum which was washed with tap water to remove impurities, and dried for one day in the shade was cut finely, and added with 2L of 70%(v/v) aqueous ethanol solution. Then it was extracted for 2 hours by using a reflux extractor [Heating mantle (GLASS-COL), Flask (Corning), Condenser (Coming)], filtered with filter paper (Whatman No.2), and concentrated under the vacuum to obtain an ethanol extract of Angelica koreanum. The ethanol extract was performed by fractional extraction done three times with the addition of 250 m£ of hydrated ethyl acetate. Then, only ethyl acetate fraction was collected, and concentrated under the vacuum at 60 to 70⁰C to obtain dried extract of Angelica koreanum at a yield of 12.75 g.

Example 1 -2: Extract of Artemisia apillaries

85 g of Artemisia apillaries were extracted according to substantially the same method of Example 1-1 to obtain 1.74 g of the dried extract.

Example 1-3: Extract of Perilla frutescens

85 g of Perilla frutescens was extracted according to substantially the same method of Example 1-1 to obtain 2.45 g of the dried extract.

Example 1-4: Preparation of herbal composition The extracts of Angelica koreanum, Artemisia apillaries, and Perilla frutescens obtained in Examples 1-1 to 1-3 were mixed in a weight ratio of 5:3:2 to produce 100 g of mixture. The mixture was dissolved in 25OmL of hydrated ethyl acetate, homogenated, and concentrated under vacuum.

Example 2: Preparation of herbal composition

5Og of dried Angelica koreanum , 3Og of dried Artemisia apillaries, and 2Og of dried Perilla frutescens with a weight ratio of 5:3:2 were extracted with 2.5£ of 70% aqueous ethanol solution for 2 hours in a reflux extractor (Heating mantle [GLASS-COL], Flask [Corning], Condenser [Coming]), filtered with filter paper (Whatman No.2), and concentrated under the vacuum to obtain 24.46 g of an ethanol extract. The ethanol extract was performed by fractional extraction done three times with the addition of 250 mO, of hydrated ethyl acetate. Then, only ethyl acetate fraction was collected, and concentrated under the vacuum at 60 to 70⁰C to obtain the dried extract. Finally, the dried extract was dispersed in 5OmL of distilled water, and freeze-dried to provide a herbal composition in powder of 3.88 g.

Example 3: Preparation of herbal composition

3Og of dried Angelica koreanum, 3Og of dried Artemisia apillaries, and 3Og of dried Perilla frutescens with a weight ratio of 1 :1 :1 were extracted with 2.51 of 70% aqueous ethanol solution for 2 hours in a reflux extractor (Heating mantle [GLASS-COL], Flask [Corning], Condenser [Coming]), filtered with filter paper (Whatman No.2), and concentrated under the vacuum to obtain 21.04 g of an ethanol extract. The ethanol extract was performed by fractional extraction done three times with the addition of 250 m£ of hydrated ethyl acetate. Then, only ethyl acetate fraction was collected, and concentrated under the vacuum at 60 to 70⁰C to obtain the dried extract. Finally, the dried extract was dispersed in 5OmL of distilled water, and freeze-dried to provide a herbal composition in powder of 4.02g.

Experimental Example 1 : Anti-inflammatory effect of Angelica Koreanum extract, Artemisia capillaries extract, and Perilla frutescens extract, and herbal composition.

Raw 264.7 cells, macrophage derived from a mouse, were purchased from a

Korean cell line bank. 2 x 10⁶ cells were inoculated in 24 well plates containing DMEM media including 10% Fetal Bovine Serum (Fetal Bovine Serum 26140-079,

Gibco), 100 U/ml penicillin, and 10j«g/ml streptomycin, and incubated for 24 hours,

37 °C , at 5% CO₂, under humid conditions.

To induce NO production in Raw 264.7, the incubated cells were injected with 1μg/ml of LPS which was a component of a bacterial cell wall. Then, each composition obtained in Examples 1-1 , 1-2, 1-3, 1-4, 2 and 3 were added in experimental groups to become a concentration of 20 μg/ml, but only dimethyl sulfoxide (DMSO) was treated in a control group. The treated cells were incubated for 16 hours. The culture media were mixed with grease, left at room temperature for 10 minutes, and their absorbance were measured at a wavelength of 540 nm. The result is represented in Fig.1

Experimental Example 2: Inhibition of NO production in mouse macrophage

To investigate the anti-inflammatory activity of the herbal compositions obtained in Examples 1-4, 2 and 3, the inhibition of NO production in mouse macrophage was tested. Raw 264.7 cells, macrophage derived from a mouse, were purchased from a Korean cell line bank. 2 x 10⁶ cells were inoculated in 24 well plates containing DMEM media including 10% Fetal Bovine Serum (Fetal Bovine Serum 26140-079, Gibco), 100 U/ml penicillin, and 10 μg/ml streptomycin, and incubated for 24 hours, 37 °C , at 5% CO₂, under humid conditions.

To induce NO production in Raw 264.7, the incubated cells were injected with 1 μg/ml of LPS. Then, each composition obtained in Examples 1-4, 2 and 3 were added in test groups to become a concentration of 0.8, 4, 20μg/ml, respectively, but only DMSO was treated in a control group. The treated cells were incubated for 16 hours. The culture media were mixed with grease, left at room temperature for 10 minutes, and their absorbance were measured at a wavelength of 540 nm. The result is represented in Figs. 2A to 2C. As shown in Figs. 2A to 2C, the herbal composition of the present invention inhibited NO production in a concentration-dependent manner in the mouse macrophage Raw 264.7. In addition, the herbal compositions of Example 1-4 and Example 2 had larger anti-inflammatory activity than that of Example 3.

Experimental Example 3: Inhibition of prostagladin E₂ (PGE₂) in mouse macrophage

To investigate an anti-inflammatory activity of the herbal compositions obtained in Example 1-4, Example 2 and Example 3, the inhibition of prostagladin E₂ production in mouse macrophage was tested by using a PGE2 immunoassay kit (Cayman, NO.514010).

According to substantially the same method of Experimental Example 2, each composition obtained in Example 1-4, Example 2 and Example 3 were added in experimental groups to become a concentration of 0.8, 4, 20μg/ml, respectively, but only DMSO was treated in a control group. The treated cells were incubated for 16 hours. 50μi of culture solution, 50μi of PGE₂-AChE Tracer, and 50//4 of PGE₂

Monoclonal Antibody were added to the enzyme-linked immunoassay kit, and incubated at 4⁰C, for 18 hours. Then, each well was washed, incubated in the shade for 60 to 90 minutes with the addition of 200/^ of Ellman's reagent, and then an absorbance measurement at a wavelength of 405nm was performed. The result is shown in Figs. 3A to 3C, where the LPS label was referred to as the total amount of prostagladin E₂ produced after osteoarthritis in the control group.

As shown in Figs. 3A to 3C, the herbal composition of Example 1-4 inhibited PGE₂ production in the mouse macrophage in a concentration-dependent manner.

Experimental Example 4: Inhibition of NO production in rabbit cartilaginous tissue

To investigate the anti-inflammatory activity of the herbal compositions obtained in Examples 1-4, 2 and 3, the inhibition of NO production in rabbit cartilaginous tissue was tested. The cartilage cells were obtained by separating the cartilaginous tissue from the back leg joints of a 2 to 3 week-old New Zealand white rabbit. 1 x 10⁶ cartilage cells were inoculated in 24 well plates containing DMEM media including 10% Fetal Bovine Serum (Fetal Bovine Serum 26140-079, Gibco), 100 U/ml penicillin, and

10μg/ml streptomycin, and incubated for 5 to 6 days, 37 ⁰C, at 5% CO₂, under humid conditions. When the cartilage cells has grown to 80%, the culture media were changed with serum free DMEM. Then, the incubated cells were added with LCM. Then, each composition obtained in Examples 1-4, 2 and 3 were added in experimental groups to become a concentration of 0.8, 4, 20//g/ml, respectively, but only DMSO was treated in a control group. The treated cells were incubated for 16 hours. The culture media were mixed with grease in a ratio of 1 :1 , left at room temperature for 10 minutes, and their absorbance were measured at a wavelength of

540nm. The result is represented in Figs. 4A to 4C.

As shown in Figs. 4A to 4C, the herbal composition of Examples 1-4, 2 and 3 inhibited NO production in a concentration-dependent manner in rabbit cartilaginous tissue. Such findings suggested that the herbal composition had an inflammatory activity on osteoarthritis.

Experimental Example 5: Inhibition of prostagladin E₂ (PGE₂) in rabbit cartilaginous tissue To investigate the anti-inflammatory activity of the herbal compositions obtained in Examples 1-4, 2 and 3, the inhibition of prostagladin E₂ production in rabbit cartilaginous tissue was tested using a PGE2 immunoassay kit (Cayman, NO.514010).

According to substantially the same method as Experimental Example 4, the incubated cells were added with LCM and incubated for 20 minutes, except for a control group. Then, each composition obtained in Examples 1-4, Example 2 and Example 3 were added in test groups to become a concentration of 0.8, 4, 20μg/ml, respectively, but only DMSO was treated in the control group. The treated cells were incubated for 16 hours. δO≠ of culture solution, 50 ≠, of PGE₂-AChE Tracer, and 50 μl of PGE₂ Monoclonal Antibody were added to the enzyme-linked immunoassay kit, and incubated at 4°C, for 18 hours. Then, each well was washed, incubated in the shade for 60 to 90 minutes with the addition of 200/iC of Ellman's reagent, and then the absorbance measurement at a wavelength of 405 nm was performed. The result is shown in Figs. 5A to 5C. As shown in Figs. 5A to 5C, the herbal composition of Examples 1-4, 2 and 3 inhibited the prostagladin E₂ production in a concentration-dependent manner in rabbit cartilaginous tissue.

Experimental Example 6: Inhibition of proteoglycan decomposition in rabbit cartilaginous tissue

To investigate the protecting effect of herbal compositions obtained in Examples 1-4, 2 and 3 on articular tissue, the inhibition of proteoglycan decomposition was tested.

According to Experimental Example 3, the cartilage cells were obtained by separating the cartilaginous tissue from the back leg joints of a 2 to 3 week-old New

Zealand white rabbit. 1 x 10⁶ cartilage cells were inoculated in 24 well plates containing DMEM media including 10% Fetal Bovine Serum (Fetal Bovine Serum

26140-079, Gibco), 100 U/ml penicillin, and 10jMg/ml streptomycin, and incubated for

5 to 6 days, 37 ^°C , at 5% CO₂, under humid conditions. When the cartilage cells had grown to 80%, the culture media were changed with serum free DMEM. Then, the incubated cells were added with LCM and incubated for 20 minutes, except for the control group. Then, each composition obtained in Examples 1-4, 2 and 3 were added in experimental groups to become a concentration of 0.8, 4, 20μg/ml, respectively, but only DMSO was treated in the control group. The treated cells were incubated for 16 hours.

After inducing proteoglycan decomposition in the culture solution, an amount of Glucosaminoglycan (GAG) was analyzed by measuring the color of 1 ,9-dimethylmethylene blue dye (DMB) at a wavelength of 525nm, and standardized with chondroitin sulfate. The result is represented in Fig. 6.

As shown in Figs. 6A to 6C, the herbal composition of Examples 1-4, 2 and 3 inhibited the proteoglycan decomposition and increased proteoglycan concentration in the synovial fluid.

Experimental Example 7: Inhibition of MMP-9 activity in rabbit cartilaginous tissue

To investigate the inhibitory effect of the herbal composition obtained in Example 1-4, herbal composition on articular tissue protease MMP-9 activity was tested. According to Experimental Example 4, the cartilage cells were obtained by separating the cartilaginous tissue from the back leg joints of a 2 to 3 week-old New Zealand white rabbit. 1 x 10⁶ cartilage cells were inoculated in 24 well plates containing DMEM media including 10% Fetal Bovine Serum (Fetal Bovine Serum

26140-079, Gibco), 100 U/ml penicillin, and 10//g/ml streptomycin, and incubated for 5 to 6 days, 37 ⁰C , at 5% CO₂, under humid conditions. When the cartilage cells had grown to 80 %, the culture media were changed with serum free DMEM. Then, the incubated cells were added with LCM and incubated for 20 minutes, except for a control group. Then, composition obtained in Example 1-4 was added in experimental groups to become a concentration of 0.8, 4, 20μg/ml, respectively, but only DMSO was treated in the control group. The treated cells were incubated for 16 hours.

The supernatant was obtained by centrifuging the cultured cells and removing cell debris. The supernatant was mixed with dye, incubated at 37⁰C, for 1 hour, and then performed by SDS-PAGE with 10% Zymogram gel (Novex EC61752) at 100 V of voltage for 3 hours. The gel was washed by shaking it three times for 30 minutes, immersed in developing buffer, and developed in an incubator for 48 hours at 37 °C. Then, the gel was stained with Coomassie G blue dye, and except for band region was bleached. The band including 92kD of MMP-9 was identified to show the result in Fig. 7. As shown in Fig.7, the herbal composition of Example 1-4 inhibited MMP-9 activity in a concentration-dependent manner.

Experimental Example 8: Inhibition of causal factor of acute inflammation in rabbit cartilaginous tissue. To investigate the inhibitory effect of the herbal composition obtained in

Example 1-4 on the COX-2 synthesizing prostaglandin, western blotting was performed.

According to Experimental Example 4, rabbit cartilage cells were obtained. The culture media were removed from the cells, treated with the herbal composition of Example 1-4 and LCM, and the cells were isolated with the addition of an extracting buffer solution (0.32 M sucrose [S0389, Sigma], 0.2 M HEPES (N-2-hydroxyethylpiperazine-N-2-dthanesulfonic acid) [H3375, Sigma], 1mM EDTA (ethylenedia mintetraacetic acid) [808288, BM], 1 mM PMSF(phenylmethylsulfonyl fluoride) [P7627, Sigma], 10μg/ml Aprotinin [A1153, Sigma], and 10jUg/ml leupeptin [L0649, Sigma]) to perform the quantitative anaylsis of proteins. Then, 20μg of proteins were treated by SDS-PAGE including SDS (sodium dodecyl sulfate) and 10% polyacrylamide gel. The treated proteins were transferred to PVDF (polyvinylidene difluoride) membrane (IPVH00010, Millipore), reacted with 5% NFDM (non-fat dry milk) solution, a first antibody, and then a second antibody, and treated by a chromogenic reaction with an ECL reagent (Amersham Biosciences) to expose them to the X-ray. 2//g/ml of COX-2 (sc-1745, Santacruze), and Anti-Rabbit-lgG-HRP

(sc-2004, Santacruze) 80ng/ml were used as the first antibody, and second antibody, respectively. The result is represented in Fig. 8.

As shown in Fig. 8, the herbal composition obtained in Example 1-4 inhibited the COX-2 expression in rabbit cartilaginous tissue to the normal extent. Experimental Example 9: Inhibition of acetic acid writhing in mouse

To investigate the analgesic effect of Example 1-4 and Example 2, an acetic acid writhing test was performed according to Whittle's method (1957).

The herbal compositions of Example 1-4 and Example 2 were administered to a mouse (ICR, Institute of Cancer Research) at an amount of 100, 500, and IOOOmg per 1kg of the mouse's body weight, respectively. After 30 minutes, 0.1 ml/1 Og of 0.7% acetic acid-saline was injected to the abdominal cavity of the mouse. For 10 minutes to 20 minutes after injection, the number of writhing was observed as shown in result Table 1. The control group was treated in the same method of the test group, except for the administration of the herbal composition. Table 1]

As shown in Table 1 , the herbal compositions obtained in Example 1-4 and Example 2 reduced the writhing numbers by 20 to 60%, which suggested good analgesic activity

Experimental Example 10: Inhibition of acute inflammation in rat

To investigate the anti-inflammatory activity of herbal compositions obtained in Example 1-4 and Example 2, the inhibitory effect of the herbal compositions on foot edema was tested by treating the herbal composition in carrageenan-causing inflammation in a rat.

The herbal compositions were orally administered to a SD (Spraque-Dawley) rat starved overnight in an amount of 200, and 400mg/kg, respectively. After 1 hour,

0.1 ml of 1% carrageenan dissolved in saline solution was administered in the sole of the right back foot of the rat subcutaneously. After 5 hours, the foot edema was measured with a plethymometer at 1 hour intervals. The inhibition rate of the edema is shown in Fig. 9. The control group was not treated with oral administration.

As shown in Fig. 9, the herbal compositions obtained in Example 1-4 and Example 2 inhibited inflammation by reducing the foot edema.

Experimental Example 11 : Decomposition of reactive oxygen species

To investigate the decomposition of reactive oxygens of the herbal compositions obtained in Example 1-4 and Example 2, SOD, which protects the body by removing reactive oxygens generated with xanthine-xanthine oxidase was measured with Superoxide Dismutase Assay Kit (Cat No. 706002, Cayman) to the decomposition degree of reactive oxygens. The positive control was treated with

Joins® (SK PHARMA CO., LTD.), and the negative control was not treated with the herbal composition.

Each well containing 200μβ of redial detector was added with 10μ£ of the herbal composition obtained in Example 1-4 and 10μ£ of SOD standard, treated with xanthine-xanthine oxidase to generate reactive oxygens, and incubated at room temperature for 20 minutes. The absorbance of the product was measured with a spectophotometer at 540nm. The decomposition degree was shown by analyzing the slope of the tangent line in Table 2. In Table 2, the SOD value was measured by using 0.7 value measured in the negative control group as a revised value. As shown in Table 2, the herbal compositions obtained in Example 1-4 and Example 2 had good decomposition activity of reactive oxygens. [Table 2]

Experimental example 12: Formalin test

To investigate the analgesic effect of the herbal compositions obtained in Example 1-4 and Example 2, the formalin test was carried out. The control group was not treated with the administration of herbal composition.

The mouse was administered the herbal composition on sole of its right back food subcutaneously, and observed in an acrylic observation chamber (20cm high,

20cm diameter) for 40 minutes. The mouse's behavior, such as a lick or beat of the sole of its foot were recorded in Fig. 10. The first 5 to 40 minutes is referred to as the 1^st phase, and the remnant 20 minutes is referred to as the 2^nd phase.

As show in Fig. 10, the herbal composition of Example 1-4 and Example 2 have excellent analgesic effects.

Experimental Example 13: Cartilage cell growth

To investigate the effect of the herbal composition obtained in Example 2 on the regeneration of cartilage cell, the cartilaginous tissue of both the back legs of a New Zealand white rabbit were operated on for a Anteior Crucuate Ligament Tansection (ACLT). After three days, the rabbit was exercised for 2 weeks in a 5 x 5m³ size chamber continuously to induce the arthritis, and then was administered the herbal composition obtained in Example 2 for 1 month. After the experiment ended, the cartilage was taken from the rabbit to compare the cartilage cell growth. The control group contained the cartilage tissue without the treatment of the herbal composition. As shown in Fig. 11 , the articular cartilage taken from the rabbit with treatment of the herbal composition obtained in Example 2 had a smooth surface of cartilage region. On the other hand, the surface of cartilaginous tissue in the control group was rough and irregularly shaped. Such a result indicated that the herbal composition of Example 2 had excellent regenerative effects on damaged cartilage cells.

Experimental Example 14: Total amount of articular synovial fluid in animal model of osteoarthritis

It has been reported that the amount of synovial fluid depends on the seriousness of osteoarthritis. After osteoarthritis was induced according to the method of Experimental Example 13, the total amount of synovial fluid was measured in 2 weeks and 4 weeks after oral administration to determine the amount of synovial fluid. The control group was treated in the same method as the test group but without the oral administration of the herbal composition.

The amount of synovial fluid was measured by determining Ca²⁺concentration in the synovial fluid according to Arsenazo III complexion method) [Micaylova V . Et al., Anal. Chim. Acta, 53(194), 1971], and total amount of synovial fluid was calculated with the Donnan equilibrium equation. 0.01 mL of synovial fluid was mixed with 1 mL of Arsenazo III reagent (Sigma), incubated at a room temperature for 5 minutes, and then its absorbance was measured at a wavelength of 600nm. The result of this Experimental Example is shown in Fig. 12 and Table 3.

As shown in Fig. 12 and Table 3, the synovial fluid of the control group increased to 0.76mL, but the synovial fluid of the test group was 0.29mL Thus, the increase of synovial fluid was inhibited in the rabbit with the oral administration of the herbal composition of Example 2 compared to the levels of the control group. [Table 3]

Experimental Example 15: Measurement of proteoglycan concentration in synovial fluid in animal model of osteoarthritis

In order to investigate the protective effect of the herbal composition, articular synovial fluid taken from the test group and control group obtained in Experimental Example 14 was used for measuring proteoglycan concentration in synovial fluid according to Experimental Example 6. The result of this Experimental example was represented in Fig. 13 and Table 4.

As shown in Fig. 13 and Table 4, the proteoglycan concentration of the control group was

after an increase of 60.12%, but that of the test group was only LZZμglμi from an increase of 35.98%. Such a result indicated that the herbal composition offered good protection of the joints. [Table 4]

Experimental Example 16: Measurement of total proteins content in synovial fluid in animal model of osteoarthritis

As osteoarthritis progresses, whole proteins are released into synovial fluid due to cartilage rupture. To investigate the inhibition of osteoarthritis, the total protein amount in synovial fluid was measured.

Articular synovial fluid taken from the test group and control group obtained in Experimental Example 14 was used and the total protein amount was obtained by measuring absorbance at 595nm according to the Bradford method. The result of this Experimental Example is indicated in Fig. 14 and Table 5.

As shown in Fig. 14 and Table 5, the total protein amount of the control group was 52.71 μg/μβ which had increased by 24.99% on the basis of 25.67 μg/μl being the normal range, but that of the test group was 38.62//g/μβ which had decreased by 26.68% based on the control group figures. [Table 5]

Experimental Example 17: Measurement of prostagladin E₂ content in synovial fluid of animal mode of osteoarthritis In order to investigate the anti-inflammatory activity of the herbal composition, articular synovial fluid taken from the test group and control group obtained in Experimental Example 14 was used for measuring prostagladin E₂ concentration in synovial fluid according to the same method of Experimental Example 5. The result of this Experimental Example Is indicated in Fig. 15 and Table 6.

As shown in Fig. 15 and Table 6, prostagladin E₂ concentration in the synovial fluid of the control group had changed from O.QδμglμH to 2Λμglμi with an increase of 221.78%. However, the prostagladin E₂ concentration in the synovial fluid of the test group had increased by 25.21%. Thus, the test group had a greater inhibition effect than the control group. Fable 6]

Experimental Example 18: Measurement of cell distribution in synovial fluid membrane and Safranin-O staining result

After inducing osteoarthritis according to the method of Experimental Example 13, the herbal composition of Example 2 was orally administered to a rabbit in an amount of 200 mg/kg for 1 month. Then, the digital thighbone was excised from the rabbit, fixed in 4% formalin (Sigma, F0507) for more than 24 hours, cut into a specimen with a 5mm thickness, and decalcified with 5% niric acid for 24 hours to prepare for the paraffin block. 4μm of the specimen was stained with Sapranin-0

(Sigma S2255) according to histochemistry analysis to investigate the cell distribution in the synovial fluid membrane and surface. The results of this Experimental Example is indicated in Fig. 16. The digital thighbone excised from the rabbit without treatment of the herbal composition was used as the control group. As shown in Fig. 16, the test group whom had been orally administered with the herbal composition of Example 2 for 4 weeks had a blue stained region overlapping the red stained region of osteoarthritis, compared to that of the control group. Thus, the result indicated that the herbal composition had the effects of regenerative activity of cartilage.

Preparation 1 : Tablet

2100mg of the herbal composition obtained in Example 2, 90mg of corn starch, 180mg of lactose, 18mg of L-hydroxylpropylcellulose, 5mg of polyvinylpyrrolidone and a remnant of ethano were mixed homogeneously, granulated by wet granulation, and performed by tableting technology with the addition of 1.8mg of magnesium stearate resulting in the weight of 400 mg per 1 tablet.

Preparation 2: Soft capsule 100mg of the herbal composition obtained in Example 2, 180mg of soy oil, 40 mg of yellow wax, 128mg of hydrogenated coconut palm oil, 20.5 mg of phopholipid soy bean , 212 mg of gellatin, 50mg of glycerin (density of 1.24), 76mg of d-sorbitol, 0.54mg of methyl p-hydroxybenzoate, 0.90mg of propyl p-hydroxybenzoate, 0.56mg of methylvanillin and a suitable amount of yellow pigment no. 203 to produce soft capsules according to the general requirements for pharmaceutical preparations were required.

Preparation 3: Capsule

100mg of the herbal composition obtained in Example 2, 83mg of corn starch, 175mg of lactose and 2mg of magnesium stearate were mixed homogeously, and were filled in quantities of 360mg into a capsule.

Preparation 4: health care food and beverage Health care foods and beverages were prepared including the herbal composition obtained in Example 2 as follow.

Preparation 4-1: Heatlh care food 1.OOOmg of the herbal composition obtained in Example 2, 70j«g of Vitamin

A acetate, 1mg of Vitamin E, 0.13mg of Vitamin B₁, 0.15mg of Vitamin B₂, 0.5mg of Vitamin B₆, 0.2μg of Vitamin B₁₂, 10mg of Vitamin C, 10μg of biotin, 1.7mg of niacin,

50μg of folic acid, 0.5mg of calcium pantothenate, 1.75mg of ferrous sulfate (FeS04 ),

0.82mg of zinc oxide, 25.3mg of magnesium carbonate, 15mg of potassium phosphate monobasic, 55mg of potassium phosphate dibasic, 90mg of potassium citrate, 100mg of calcium carbonate, and 24.8mg of magnesium chloride were prepared according to the general method of producing health care foods.

The mixed ratio of vitamins and minerals represented are preferable, but they can be modified. The food can be prepared by mixing and granulating according to the general method.

Preparation 4-2 : Health care beverage

100mg of the herbal composition obtained in Example 2, 15g of Vitamin C,

100g of Vitamin E(powder), 19.75g of ferrous lactate,3.5g of niacin, 3.5g of zinc oxide, 0.2g of Vitamin A, 0.25g of Vitamin B1 , 0.3g of Vitamin B2, and water were mixed and prepared to produce health care beverages according to the general method.

According to the general method of preparing health care beverages, the components were mixed, agitated for 1 hour at 85 °C with heating, filtered, and collected to pour into 2L of a sterilized vessel. The vessel was stored in a refrigerator, and then used to prepare the health care beverage.

The above composition is represented as a desirable example, but can be modified depending on the person in need, county, usage, taste, and the like.

Preparation 4-3: Chewing gum The chewing gum was prepared by mixing 0.24 ~ 0.64% of the herbal composition obtained in Example 2, 20% gum base, between 76.36 ~ 76.76% of sugar, 1 % of fruit flavor and 2% water according to the general method of preparing chewing gum.

Preparation 4-4: Ice cream

The ice cream was prepared by using 0.24 ~ 0.64% of the herbal composition obtained in Example 2, 10.0% milk fat, 10.8% fat free solid, 12.0% sugar, 3.0% starch syrup, 0.5% emulsion stabilizer (span), 0.15% flavor (strawberry), and between 62.91 ~ 63.31% water according to the general method of preparing ice-cream.

Preparation 4-5: Beverage

The beverage was prepared by using 0.48 ~ 1.28mg of the herbal composition obtained in Example 2, 522mg of honey, 5mg of thioctanamide, 10mg of niacin, 3mg of sodium riboflavin chloride, 2mg of pyridoxine chlorate,30mg of inositol,

50mg of orotic acid, and 200mϋ of water according to the general method of preparing beverages.

The herbal composition of the present invention comprising of Angelica koreanum extract, Artemisia apillaries extract, and Perilla frutescens extract has an efficacy in anti-inflammatory activity, anti-inflammatory and analgesic activities, and inhibitory activities on articular tissue protease, inflammatory enzymes, foot edema, increase of articular synovial fluid, and increase of whole proteins of synovial fluid, and the like, and thus can be used as an agent of regenerating cartilaginous tissue and an agent of treating osteoarthritis. In addition, the composition can be used as a pharmaceutical composition or food composition.

Claims

WHAT IS CLAIMED IS:

1. A composition for regenerating cartilaginous tissue comprising an Angelica koreanum extract which is prepared by extracting with ethanol, distilled water, or a mixture thereof, an Artemisia apillaries extract which is prepared by extracting with ethanol, distilled water, or a mixture thereof, and a Perilla frutescens extract which is prepared by extracting with ethanol, distilled water, or a mixture thereof.

2. The composition according to Claim 1 , wherein the composition comprises 5 to 50 parts by weight of the Angelica koreanum extract, 3 to 30 parts by weight of the Artemisia capillaries extract, and 2 to 20 parts by weight of the extract of Perilla frutescens.

3. The composition according to Claim 1 , wherein the composition was prepared by extracting a plant mixture or each plant of Angelica koreanum, Artemisia capillaries, and the Perilla frutescens, with ethanol, distilled water, or a mixture thereof, and further performing a fractional extraction with ethyl acetate to produce ethyl acetate fraction.

4. The composition according to Claim 1 , wherein the composition inhibits decomposition of proteoglycan.

5. The composition according to Claim 1, wherein the composition is in a dosage form selected from the group consisting of oral tablets, oral soft capsules, ointments, injections, and a transdermal dosage form.

6. A composition for treating osteoarthritis comprising 5 to 50 parts by weight of Angelica koreanum extract which is prepared by extracting with ethanol, distilled water, or a mixture thereof, 3 to 30 parts by weight of Artemisia apillaries extract which is prepared by extracting with ethanol, distilled water, or a mixture thereof, and 2 to 20 parts by weight of Perilla frutescens extract by extracting with ethanol, distilled water, or a mixture thereof.

7. The composition according to Claim 6, wherein the composition was prepared by extracting a plant mixture or each plant of Angelica Koreanum, Artemisia capillaries, and the Perilla frutescens, with ethanol, distilled water, or a mixture thereof, and further performing a fractional extraction with ethyl acetate to produce ethyl acetate fraction.

8. The composition according to Claim 6, wherein the composition reduces the increased blood amount of proteoglycan, prostaglandin, matrix metalloprotease-9 (MMP-9), and cyclooxygenase-2 (COX-2) which are markers of osteoarthritis.

9. The composition according to Claim 6, wherein the composition is in a dosage form selected from a group consisting of oral tablets, oral soft capsules, ointments, injections, and a transdermal dosage form.

10. A method of preparing a composition for regenerating cartilaginous tissue or a composition for treating osteoarthritis comprising the steps of: extracting a plant mixture or each plant of Angelica Korean, Artemisia capillaries, and the Perilla frutescens, with at least a solvent selected from a group consisting of distilled water, C1 to C5 alcohol, ethyl acetate and a mixture thereof; and concentrating the extracts by performing a fractional extraction and separating a fraction of ethyl acetate.