KR20080055205A - Composition comprising mixed extract of aralia cordata thunb and vitex rotundifolia l. fil for preventing and treating arthritis - Google Patents

Abstract

A composition comprising the mixed extracts of Aralia cordata THUNB and Vitex rotundifolia L. fil is provided to suppress edema and pain, inhibit proteoglycan in articular cartilage cell and activity of MMP-1(matrix metalloproteinase-1) and MMP-3, and induce no cytotoxicity and morphological change, so that the composition is useful for preventing and treating arthritis. A pharmaceutical or health food composition for preventing and treating arthritis comprises 0.1-50 wt.% of the mixed extracts of Aralia cordata THUNB and Vitex rotundifolia L. fil which are prepared by extracting them with water, C1-C4 lower alcohol including methanol or ethanol, or a mixed solvent thereof, wherein the arthritis is degenerative arthritis, rheumatoid arthritis or Lupus arthritis; and the daily dosage of the mixed extracts is 0.01mg/kg-10g/kg, preferably 1 mg/kg-1g/kg.

Description

 Composition containing mixed extract of Aralia cordata THUNB and Vitex rotundifolia L. fil for preventing and treating arthritis}

      Figure 1a is a diagram confirming the inhibition of chondrocyte degradation by interleukin-1 in the treatment of the venom and Manza mixture (ACVR-T) of the present invention through the concentration of GAG (Glycosaminoglycan) in human joint medium,

      Figure 1b is a diagram showing the inhibition of chondrocyte degradation by interleukin-1 in the treatment of the venom and morphology mixture (ACVR-B) of the present invention through the concentration of GAG (Glycosaminoglycan) in the human joint medium,

Figure 2a is a diagram confirming the inhibition of chondrocyte degradation by interleukin-1 in the treatment of toxin and morphology mixture (ACVR-T) of the present invention through the concentration of GAG (Glycosaminoglycan) in the rabbit joint medium,

      Figure 2b is a diagram showing the inhibition of chondrocyte degradation by interleukin-1 in the treatment of virulence and morphology mixture (ACVR-B) of the present invention through the concentration of GAG (Glycosaminoglycan) in rabbit joint medium,

 Figure 3a is a diagram showing the inhibition of MMP-1 activity by interleukin-1 in the treatment of the venom and Manza mixture (ACVR-T) of the present invention,

       Figure 3b is a diagram showing the inhibition of MMP-1 activity by interleukin-1 in the treatment of toxin and mantrel mixture (ACVR-B) of the present invention,

Figure 4a is a diagram showing the inhibition of MMP-3 activity by interleukin-1 in the treatment of the venom and Manza mixture (ACVR-T) of the present invention,

       Figure 4b is a diagram showing the inhibition of MMP-3 activity by interleukin-1 in the treatment of the venom and Manza mixture (ACVR-B) of the present invention,

 Figure 5 is a diagram showing the measurement of the effect on the chondrocyte survival of the venom and manza mixture treatment of the present invention.

     The present invention relates to a composition for the prevention and treatment of arthritis, which contains a mixed herbal extract of venom and man form as an active ingredient.

In oriental medicine, arthritis is mainly expressed as nausea, and the clinical manifestation of nausea is mainly pain, and pain can be caused by pathological problems of blood circulation. The main external factors can be classified into wind, cold, damp, and heat.These causes the blood circulation problems such as joints and muscles, and pains. Limitations of range of motion appear. Internal factors are the major causes of the liver and kidneys, the muscles belonging to the liver, the liver presides over blood, To nourish (滋養) to regulate the blood of the liver should be smooth to prevent pathological products such as blood (瘀血). In addition, God is in charge of the bones (腎 主 조절) and regulates the human body (精), this tablet (精) functions to nourish the bone marrow and self-lubricating the skeleton. Therefore, the abnormalities of the joints are closely related to the functions of the liver and the kidneys. Therefore, the prevention and treatment of osteoarthritis in oriental medicine is aimed at more fundamental treatment based on these principles. Degenerative arthritis, one of the major osteoarthritis diseases, is chronic arthritis. It is difficult to cure with anti-inflammatory drugs that are currently used in clinical trials as intractable diseases, and it also causes systemic side effects such as digestive disorders, gastrointestinal disorders, and decreased kidney function. Long-term systemic administration in the treatment of many bone joint diseases has many problems. Therefore, the development of new drugs aimed at protecting and regenerative effects of anti-inflammatory and cartilage for the improvement and prevention of symptoms more aggressively than the symptomatic approach such as anti-inflammatory action on degenerative arthritis that increases in proportion to the aging of the population. Everything is desperate. Overseas, in addition to the development of anti-inflammatory drugs in recent years, with interest in articular cartilage protection, prevention of cartilage degradation and regeneration, joint histase inhibitors, free radical scavengers (active oxygen scavenging enzymes such as SOD) Research on conservation therapy by long-term administration of tissue components (Chondroitin, Glucosamine etc.) has been actively conducted (Badger, AM, Cook, MN, et al., Journal of Pharmacology and Experimental Therapeutics 290 , pp 587-593, 1999)

On the other hand, in the Asian countries such as Korea, China, and Japan, the disease treatment agent has been used for thousands of years, and its efficacy has been proven, and the secretariat has been used in the private sector. Based on the selection of herbal medicines that can be developed based on the present invention, and the mechanism of the research through more rigorous basic research, a new concept of arthritis therapeutic herbal medicine was developed to complete the present invention.

Single life (獨 活, Aralia cordata THUNB.) is a perennial herb belonging to the family Araliaceae, up to 1.2 m high, with short hairs around the stem. The leaves are alternated with the leaves of 3 to 5 leaves, with the teeth on the edges of the leaves. The flower is light green, and the female flower and the male flower bloom in one branch, and in July-August, the inflorescence blooms at the end of the branch. Fruit is berry and ripens in October in black. It is often planted for use as a medicine. In the spring, the poisonous poison is used to treat migraines in the spring and autumn by cutting off the rhizome and roots and peeling the sun. As soon as the roots are dried in the sun, the fragrance will disappear, so it is better to dry them in a well-ventilated shade and dry them in the sun. The taste is hot and the nature is warm and nontoxic, fever, analgesic, jingyeong, anti-inflammatory, blood coagulation promoting action, cardiac action, coercive action. In the private sector, outposts are used as antipyretics, diuretics and analgesics. Root is used as a medicine. It is effective for dehumidification and tongjitong, so it is used to prescribe nasal pain, neuralgia, headache, and seizure sequelae. Roots contain essential oils, and essential oils include limonen, sabnene, alpha-pinene, gamma-terpinene and myrcene. , Humulene (α-caryophyllene), alpha-copaene (α-copaene) and terpinene-4-ol (terpinene-4-ol) (information and synergy, metabolism of Dohaeyang medicine, Yeonglimsa , p435, 1998).

Toxicity inhibits COX, a major inducer of arthritis, and has been reported to have therapeutic effects on analgesia, hypothermia, and muscle locomotion (Dang NH, et al., Inhibitory constituents against cyclooxygenases from Aralia cordata Thunb. Arch Pharm Res. , 28 (1) , pp. 28-33, 2005 Jan).

A full sentence ( Vitex rotundifolia L. fil) is a larva of the genus Verbena, a deciduous shrub that grows obliquely to the side, 30-60 cm high, with gray white fine hairs. The flowers are purple and run densely in the shape of a conical inflorescence 4-7 cm long. Leaves are round or obovate, wedge-shaped, blunt or round at the end, saw-toothed and intact, with purple flowers blooming in July-September. Corolla is purple, anther is purple, and fruit is nuclear, spherical, mature in September-October. Pick the ripe fruit in autumn and dry it in the sun. Use it as it is soaked or roasted and take 6-9g per day in the form of decoction, pills, and powdered medicine. Examples of taking a combination of chrysanthemum, windproof, etc. to cure a severe headache headache, the combination of ancient bones and cheongung to treat headache. The combination of chrysanthemum defects, etc. to treat the symptoms of red eyes, painful eyes and dizziness and tears caused by the satire. Cautions should be avoided for people with anemia and weak heads. In the private sector, it has a sedative effect and stops pain, so it is effective for headaches caused by neuralgia and hypertension. It is also known to calm down the body's temperature and relieve heat. It has also been reported that all types also contribute to anti-cancer effects (Kobayakawa J, Sato-Nishimori F. G2-M arrest and antimitotic activity mediated by casticin, a flavonoid isolated from Vitics Fructus (Vitex rotundifolia Linne fil. Cancer Lett. 2004 May 10 : 208 (1): 59-64).

 The inventors of the present invention tried to use more scientifically the manipulator having an effective decoction, sedative effect and pain relief effect such as antipyretic action, analgesic action, myrrhosis, anti-inflammatory action, blood coagulation promoting action, cardiovascular action, coercive action , Toxic and morphogenic mixtures confirmed the analgesic inhibitory effects, and the cartilage protective effects such as inhibition of proteoglycan release from cartilage tissues and the inhibition of MMP-1 and MMP-3 activity, cartilage degrading enzymes, to treat and prevent arthritis The present invention has been completed by confirming that it can be usefully used as a composition.

It is an object of the present invention to provide a pharmaceutical composition and health functional food for preventing and treating arthritis, containing as an active ingredient a mixed herbal extract of venom and mansiform, showing cartilage protection effect and pain relief effect.

     In order to achieve the above object, there is provided a pharmaceutical composition for the prevention and treatment of arthritis, containing the mixed herbal extracts of venom and mansip as an active ingredient.

      In addition, the present invention provides a dietary supplement for the prevention and improvement of arthritis containing the mixed herbal extracts of venom and mangyeong as an active ingredient.

      An extract as defined herein means an extract available in water including purified water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

      The arthritis includes at least one disease selected from degenerative arthritis, rheumatoid arthritis, lupus arthritis, preferably degenerative arthritis, rheumatoid arthritis.

      Hereinafter, the present invention will be described in detail.

      Mixed herbal extract of the present invention can be prepared as follows.

After dryness and grinding of the toxin and mantrel of the present invention, about 1 to 20 times the weight of the dried sample, preferably about 5 to 15 times the amount of C ₁ to C ₄ , such as water, ethanol, methanol, etc. Alcohol or a mixed solvent of water and ethanol having a mixing ratio (kg / L) of about 1: 0.1 to 1:10, preferably 1: 0.2 to 1: 5, at a temperature of about 0.5 to 20 hours, preferably 1 Extraction method obtained by stirring, extracting hot water, cold needle extraction, reflux cooling, or ultrasonic extraction for 10 hours, preferably extracting hot water after filtration, concentration under reduced pressure, or drying, is available in polar solvents. Can be obtained.

In addition, a conventional fractionation process can also be carried out (Harborne JB, Phytochemical methods: A guide to modern). techniques of plant analysis , 3 rd Ed . , pp 6-7, 1998).

Mixed herbal extracts obtained by the method described above are effective in inhibiting edema reaction, analgesic inhibitory effect, and inhibiting the expression of MMP-1 and MMP-3 which are proteoglycans and cartilage tissue degrading enzymes in articular chondrocytes. By having a cartilage cell protective effect that does not cause cytotoxicity and morphological changes, it was confirmed that it has an effective and safe prevention and treatment of arthritis.

      The present invention provides a pharmaceutical composition for the prevention and treatment of arthritis, which contains a mixed herbal extract of venom and mansip obtained by the production method as an active ingredient.

      The composition for preventing and treating arthritis diseases of the present invention comprises the mixed herbal extract in an amount of 0.1 to 50% by weight based on the total weight of the composition.

      However, the composition as described above is not necessarily limited thereto, and may vary according to the condition of the patient and the type and extent of the disease.

      Compositions comprising a mixed herbal extract of the active and full form of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

      Compositions comprising extracts according to the invention are formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. The carriers, excipients and diluents that may be used in the composition comprising the extract may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin , Calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

     Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.01 mg / kg to 10 g / kg, preferably 1 mg / kg to 1 g / kg per day. Administration may be administered once a day or may be divided several times. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

      The composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

 The present invention provides a dietary supplement for the prevention and improvement of arthritis, including a mixed herbal extract and a food supplement acceptable food supplements of the active and full form showing the effect of preventing and improving arthritis.

      The composition containing the extract of the present invention may be used in various ways, such as drugs, foods and drinks for the prevention and improvement of arthritis. Examples of the foods to which the mixed herbal extracts of the present invention and the full form of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and powders, granules, tablets, and capsules. Or in the form of a beverage.

     The mixed herbal extract itself of the present invention and the full form of the present invention has little toxicity and side effects, so it is a drug that can be used with confidence even for prolonged administration for prophylactic purposes.

     The extract of the present invention may be added to food or beverage for the purpose of preventing and improving arthritis. At this time, the amount of the extract in the food or beverage is generally added to the health food composition of the present invention to 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 10 g based on 100 ml, preferably Can be added in a ratio of 0.3 to 1 g.

     In addition to containing the extract as an essential ingredient in the indicated proportions, the health beverage composition of the present invention has no particular limitation on the liquid component, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates are conventional monosaccharides such as disaccharides such as glucose and fructose, such as maltose, sucrose and the like, and polysaccharides such as dextrin, cyclodextrin and the like. Sugars and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

      In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

       Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

 However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

Example  One. Poisonous  And Man  Mixture manufacturing

1-1. Poisonous  And Full of life  Preparation of 50% Ethanol Mixed Solvent Soluble Extract

      Cut the venom and manzas from Kyunghee Medical Center into about 0.5cm each, mix 250g evenly, add 2ℓ of 50% (v / v) ethanol and extract it under reflux for 6 hours while stirring well. It was. The filtrate was collected and collected. The residue was added to 1.5 L of 30% (v / v) ethanol solution and re-extracted for 3 hours. The filtrates were combined and concentrated to 1 L. The mixture was freeze-dried and 84.5g It used as the sample of the following experiment example (it calls it ACVR-T hereafter).

1-2. Preparation of Butanol Soluble Extracts of Toxic and Mantis

ACVR-T 84.5g obtained in Example 1-1 was fractionated by adding 500 ml of butanol and then evaporating butanol with a rotary evaporator to obtain 2.45 g of a butanol soluble extract. (Hereinafter referred to as ACVR-B)

Experimental Example  1. Measurement of analgesic activity

1-1. Foot pressure  Analgesia experiment ( Paw pressure analgesia test )

In order to determine the analgesic effect of ACVR-T and ACVR-B obtained in Examples 1-1 and 1-2, the paw pressure analgesia test was performed using the method described in the literature as follows (Khasar SG et al., Pain , 116 (1-2), pp79-86, 2005).

As a test animal, a male SD rat (central test animal, Japan) having a body weight of 200 g was used after being purified for several days. Oral administration of ACVR-T, ACVR-B (400 mg / kg) and the positive control COX-2 inhibitor celecoxib (100 mg / kg, Sigma, USA) obtained in Examples 1-1 and 1-2 above After 24 hours, 2% carrageenan (Sigma, USA) was subcutaneously administered to the left Fuji and analgesometer (Stoelting, USA) was used to measure the weight until pain avoiding response, and the weight of the negative control group. Based on the effect of celecoxib as 100, the inhibition rate was converted, and the results are shown in Table 1 below.

division Concentration (mg / kg) Edema inhibition rate (%) ACVR-T 400 113.6 ACVR-B 400 125.8 Celecoxib 100 100

Experimental results, as shown in Table 1 above, the anti-inflammatory effect by inhibiting the edema of the foot induced in mice in the mixed herbal extracts of the present invention and full form of the present invention than celecoxib, a nonsteroidal anti-inflammatory analgesic It was confirmed that the excellent.

1-2. Formalin analgesic experiment ( Formalin analgesia test )

In order to determine the analgesic effect of ACVR-T and ACVR-B obtained in Examples 1-1 and 1-2, a formalin animal model experiment was performed using the method described in the literature as follows (Fazli-Tabaei S et al. ., Behav . Pharmacol ., 16, pp 613-619, 2005).

As a test animal, male ICR mice (central test animal, Japan) having a body weight of 20 to 25 g were purified for several days, and 10 animals were used in each group. Oral administration of ACVR-T, ACVR-B (400 mg / kg) and the positive control COX-2 inhibitor celecoxib (100 mg / kg, Sigma, USA) obtained in Examples 1-1 and 1-2 above After 24 hours, 10% formalin solution (Sigma, USA) was subcutaneously administered to the left Fuji, and the time of licking the bottom of the Fuji was measured, and the inhibition rate was converted to the effect of celecoxib at 100, and the results are shown in Table 2 below. Shown in

division Concentration (mg / kg) % Pain inhibition ACVR-T 400 1st 90.4 2nd 110.1 ACVR-B 400 1st 106.3 2nd 110.2 Celecoxib 100 1st 100 2nd 100

 As a result, as shown in Table 2, the pain inhibitory effect in the mixed herbal extracts of the venom and manipulator of the present invention was similar to the celecoxib, a nonsteroidal anti-inflammatory analgesic.

1-3. Torsion pain test ( Writhing analgesia test )

In order to determine the analgesic effect of ACVR-T and ACVR-B obtained in Examples 1-1 and 1-2, the acetic acid-induced analgesic test was performed using the method described in the literature as follows (BA Whittle, Brit . J.). . Pharmacol., 22, pp246-253, 1964).

As male animals, 10 male ICR mice (20-25 g body weight) were used in each group. After oral administration of ACVR-T and ACVR-B obtained in Examples 1-1 and 1-2, pain was induced by intraperitoneal administration of 0.7% acetic acid (v / v in DW) 1 hour later. The animals were observed for 10 minutes from the next 5 minutes and recorded the number of writhing, and then the inhibition rate was converted to the number of negative controls as celecoxib 100, and the results are shown in Table 3 below.

division Concentration (mg / kg) Warp Suppression Rate (%) ACVR-T 400 116.1 ACVR-B 400 192.1 Celecoxib 100 100

Experimental results, as shown in Table 3, showed an excellent analgesic effect in the mixed herbal extracts of the poisonous and full-form of the present invention, the pain suppression effect of the mixed herbal extracts of the full-lived and full-formed type When compared with the sieve was confirmed to be superior.

Experimental Example  2. Measurement of cartilage protection effect

If cartilage-producing elements such as proteoglycans and collagen cannot be produced quickly, cartilage may be damaged and arthritis may occur if the cushion function of cartilage is lost. Articular cartilage consists of water (70-80%), collagen (10-15%), proteoglycans (5-10%), and chondrocytes. Proteoglycans contain several glycos in a single row of core proteins. Amino glycan (GAG; glycosaminoglycan) is attached to the structure.

In order to confirm the effect of proteoglycan degradation, MMP-1, and MMP-3 activity inhibition of mixed herbal extracts of the present invention, the culture of human and rabbit joint tissues described in the literature was conducted in the following manner ( Involvement of MMP-1 and MMP-3 in Collagen Degradation Induced by IL-1 in Rabbit Cartilage Explant Culture.LIFE SCIENCES 1998; VOL 62).

2-1. Experiment preparation

      Human articular cartilage was used to receive samples from patients undergoing artificial joint surgery (Kyung Hee Medical Center Orthopedic Surgery). Chondrocytes were isolated from the joints of 2 week old rabbits (Newzeland White Rabbit, Samtako Korea, Korea).

2-1-1. Cartilage Tissue Culture

5% fetal bovine serum (FBS, GIBCO) was inactivated by heat treatment of the articular cartilage of the human and rabbit obtained by exposing the surface of the joint through surgery under sterile conditions and taking approximately 200-220 mg of the joint surface tissue. BRL, USA) and Penicillin-Streptomycin 100 soaked in a basic medium (DMEM; Dulbeco's modified eagle's medium, GIBCO BRL, USA). The joint tissues were washed several times with the medium and then moist 95% CO ₂ at 37 ° C. Cultured in the incubator. After 1-2 days, the medium was replaced with basal medium containing 5% fetal calf serum inactivated by heat treatment, 10 mM HEPES, and 100 units / ml of penicillin-streptomycin, and 30 mg of joint tissue was placed in a 48-well plate. Moved. After incubation for 1 hour, 5 ng / μl of interleukin-1α (IL-1α, R & D system, USA) was added to the medium to induce inflammation, and ACRV-T and ACRV- obtained in Examples 1-1 and 1-2 above. B a 20uM to 0.4, 0.8, 2 ㎎ / ㎖ and NS398, Aralia cordata (Aralia cordata THUNB.) 0.8 mg / mL were incubated at 37 ° C. after treatment. After incubation for 3 days, the supernatant was collected, and 0.4, 0.8, 2 mg / ml of NS398 and 20 uM of ACRV-T and ACRV-B obtained in Examples 1-1 and 1-2, and Aralia cordata THUNB.) The culture solution was collected at 3, 7, 14, and 28 days of incubation for 25 days by exchange with a medium containing 0.8 mg / ml, which was used as a sample of the following experiment while storing at -20 ° C.

2-2. Determination of Glycosaminoglycan (GAG) Degradation

Degradation of glycosaminoglycans (GAG) constituting proteoglycans using the methods described in the literature to determine the protective effect of ACRV-T and ACRV-B obtained in Examples 1-1 and 1-2 above Experiments to determine the extent were performed as follows (French, MM, et al., Ann. Biomed. Eng. 32, pp 50-56, 2004).

Glycosaminoglycan (GAG) levels in the medium were measured by the amount of polyanionic material reacted with 1,9-dimethylmethylene blue, and chondroitin sulfate was used as a standard. , NS398 as positive control, independent AC ( Aralia cordata THUNB.). Spectroscopic GAG amount was measured at 540 nm, and the inhibition rate was calculated based on the amount of GAG degradation induced by interleukin 1α, and is shown in FIG. 1.

     As a result, as shown in Figure 1, the ACVR-T of Example 1-1 in the degree of GAG degradation in human joint tissue medium significantly confirmed that inhibiting chondrocyte degradation at 0.4, 0.8, 2mg / ml Could. The NS398 treatment group, a COX-2 inhibitor used as a positive control, did not inhibit GAG degradation at 20 uM, but the A.C. treatment group inhibited GAG degradation at 800 mg / ml.

     ACVR-B of Example 1-2 was found to significantly inhibit chondrocyte degradation at 0.8 mg / ml, NS398 positive control group did not inhibit the activity of MMP-1 and also inhibited toxin (AC) I couldn't. Therefore, it was confirmed that the ACVR-T and ACVR-B of Examples 1-1 and 1-2 had superior cartilage protection effect than NS398.

     As a result, as shown in Figure 2, ACVR-T and ACVR-B obtained in Examples 1-1 and 1-2 in the degree of GAG degradation in rabbit joint tissue medium cartilage at 0.4, 0.8, 2mg / ml It was confirmed that significantly inhibit the cell degradation. The NS398 treated group, which was used as a positive control group, did not inhibit GAG degradation at 20 uM, but the poisoned (A.C.) treated group inhibited GAG degradation at 800 mg / ml.

     Therefore, ACVR-T and ACVR-B were found to have a superior cartilage protection effect than NS398.

2-3. MMP - 1 and MMP -3 activity inhibition test

Matrix metalloproteinase (MMP) is a kind of proteolytic enzyme that destroys proteins in cartilage tissues. In addition to rheumatoid arthritis, osteoporosis destroys cartilage tissue and exacerbates arthritis. Of particular importance (Nagase, H., and Woessner, JF, Jr Marcel Dekker, NY., Pp159-185, 1993).

The mixed herbal extract of Toxic and Mannform obtained in Example 1 was used to examine the effect of inhibiting MMP production using rabbit cartilage explants cultures of Experimental Example 2-1-1.

MMP activity levels in the media were measured using the ELISA kit (Biomol Research Lab., Inc., PA, USA) according to the manufacturer's instructions. In brief, MMP activity is thiopeptolide (Ac) as a colorimetric substrate cut by stromyelin-1 / MMP-3 and collagenase-3 / MMP-13. -Pro-Leu-Gly- [2-mercapto-4-methyl-pentanoly] -Leu-Gly-OC ₂ H ₅ ). In order to measure the protelytic activity, each sample was dispensed with enzyme in a 96-well plate at 25 μL, incubated at 37 ° C. for 1 hour, and measured at 450 nm. MMP-1 and MMP-3 activity of each sample was measured as% of MMP contained in each medium well.

As a result, as shown in Figure 3 below, the toxin and mantrel mixture of the present invention is characterized by the activity of interleukin-1α-induced protease MMP-1 0.4, 0.8, 2 mg / ml of ACVR-T and ACVR-B Significantly inhibited in the treatment group. In addition, the NS398 treatment group, a COX-2 inhibitor used as a positive control group, did not inhibit the activity of MMP-1 and significantly inhibited toxin (A.C.). Therefore, it was confirmed that the mixed herbal extracts of the venom and mangyeong have more excellent effects than the NS-398.

Experimental results, as shown in Figure 4 below, the mixed herbal extracts of the present invention and the active agent of the present invention, the activity of interleukin-1α-induced protease MMP-3 0.4, 0.8, 2 of ACVR-T and ACVR-B significantly inhibited in the mg / ml treatment group. In addition, the NS398 treatment group, a COX-2 inhibitor used as a positive control group, significantly inhibited the activity of MMP-1 and significantly inhibited toxin (A.C.). Therefore, it was confirmed that the mixed herbal extracts of venom and mantaform have an excellent effect on the protection of articular cartilage.

2-4. Cytotoxicity Measurement

In order to determine the effect of the mixed herbal extracts of the present invention and the full form of the present invention on the viability of chondrocyte, one of the chondrocytes, experiments were performed by MTT assay using the cells of Experimental Example 2-1-1. .

10ul of CCK-8 solution was dispensed into the plate incubated for 1 day, incubated for 2 hours at 37 ° C, and then absorbance was measured at 450 nm (possible to 650 nm).

As a result of the experiment, as shown in Figure 5, the mixed herbal extracts of the venom and mansiform of the present invention does not affect the chondrocyte survival even in the culture solution for 1 day after the high concentration (0.1mg / ml), cells in the chondrocytes It did not show toxicity and could be confirmed as a safe composition.

      Hereinafter, a preparation example of a composition containing an extract of the present invention will be described, but the present invention is not intended to be limited thereto but only to be described in detail.

Formulation Example 1 Preparation of Powder

Mixed Herbal Herb Extract 20 mg

Lactose 100 mg

Talc 10 mg

    The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

10 mg mixed herbal extract

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

   After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

10 mg mixed herbal extract

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

    According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

10 mg mixed herbal extract

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _4, 12H ₂ O 26 mg

     According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation example  5. Liquid  Produce

Mixed Herbal Herb Extract 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

     According to the conventional method of preparing a liquid solution, each component is added and dissolved in purified water, lemon flavor is added to the mixture, and then the above ingredients are mixed, purified water is added to adjust the total amount to 100 ml, and then filled in a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Healthy Food

Mixed Herbal Extract 1000mg of Toxic and Mantis

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

     Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding 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 preparing a health food composition according to a conventional method.

Formulation example  7. Manufacture of health drinks

1000 mg of mixed extract of venom and sperm

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

    After mixing the above components in accordance with a conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

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

The mixed herbal extract of the present invention and the full form of the present invention is excellent in inhibiting edema in carrageenan-induced animal model, and has an effective analgesic inhibitory effect on analgesia induced by formalin or acetone, and inhibits proteoglycan in articular chondrocytes, MMP- 1 and MMP-3 activity inhibitory effect, does not cause cytotoxicity and morphological changes can be usefully used as an effective and safe composition for preventing and treating arthritis.

Claims (6)

Single Bow ( Aralia cordata THUNB.) And Man-shaped ( Vitex A pharmaceutical composition for the prevention and treatment of arthritis, which contains a mixed herbal extract of rotundifolia L. fil) as an active ingredient.  The pharmaceutical composition according to claim 1, wherein the extract is an extract available in a solvent selected from water including purified water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.  According to claim 1, wherein the pharmaceutical composition, characterized in that it comprises 0.1 to 50% by weight of the mixed herbal extract of the active and full form with respect to the total weight of the composition.  The pharmaceutical composition according to claim 1, wherein the arthritis is degenerative arthritis, rheumatoid arthritis, or lupus arthritis. Single Bow ( Aralia cordata THUNB.) And Man-shaped ( Vitex Health functional food for the prevention and improvement of arthritis containing mixed herbal extract of rotundifolia L. fil) as an active ingredient. A dietary supplement according to claim 5 which is a powder, granule, tablet, capsule or beverage.

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