KR20100019229A - Composition for treating or preventing arthritis comprising houttuynia cordata thunberg supercritical extract as an effective component - Google Patents

Abstract

PURPOSE: A composition containing Houttuynia cordata Thunberg as an active ingredient is provided to highly suppress pain, ensure anti-inflammation, and prevent and treat arthritis. CONSTITUTION: A pharmaceutical composition for preventing and treating arthritis contains Houttuynia cordata Thunberg supercritical extract as an active ingredient. The arthritis is a degenerative arthritis. The supercritical extract is obtained from roots, leaves, or stems using carbon dioxide as a solvent at 300-500 pressure and 30~80°C or 350~450pressure, and 50~70°C.

Description

Composition for treating or preventing arthritis comprising Houttuynia cordata Thunberg supercritical extract as an effective component

The present invention relates to a composition for the prevention and treatment of arthritis, containing Echocho supercritical extract as an active ingredient.

Arthritis is a representative degenerative and intractable disease, with 12% of the world's population and 70% of people over 60 years old. 4 ~ 5% of Koreans are estimated to have degenerative arthritis and about 1% of patients with rheumatoid arthritis. It is estimated that 80% of older people have degenerative arthritis.

The degenerative arthritis is a disease in which local degenerative change occurs as the articular cartilage wears off, also called osteoarthritis or osteoarthritis. Osteoarthritis is a representative degenerative disease that is closely related to aging. About 10 to 15% of the entire population suffers from osteoarthritis. In particular, about 60 to 80% of the elderly aged 65 or older suffer from osteoarthritis.

The causes of degenerative arthritis are deeply related to aging and excessive weight, and the more and more severely in women as they age. The initial symptoms are one or two joints with stiffness and pain like pain, and prolonged prolongation of the cartilage, hardening of the subchondral bone, hyperplasia of the bone around the joint, and deformation of the joint.

Degenerative arthritis can be classified as primary (primary) or secondary (secondary). First, the primary cause of primary degenerative arthritis is unknown, but it is thought to be caused by age, sex, genetic factors, obesity, etc. Primary (primary) degenerative arthritis is more common in women after middle age and more frequently in women. It is also related to family history. In the case of obesity, the incidence is about 2 times higher than that of a normal person, and it is mainly shown in a weight bearing joint. Commonly involved joints include weight-bearing and compression of the lumbar spine, hips, knees, and metatarsal joints of the foot's moss. In particular, even within the joint, a certain area, ie, the inner joint of the knee joint, is well invaded. In females, the distal joints of the resin (finger joints away from the torso) and the first carotid metacarpal joints (palm of the palm of the hand) are well invaded, and in men, hip involvement is common.

Second, secondary (secondary) degenerative arthritis can be caused by all trauma, disease and malformations that can damage joint cartilage. Congenital malformations include a lack of joint alignment, such as hip dysplasia or varus, and degenerative arthritis often occurs due to avascular necrosis, severe shock, or repeated minor trauma. In addition, many degenerative arthritis also appear in endocrine abnormalities such as acromegaly and diabetes, and metabolic diseases such as gout.

The cause of degenerative changes in articular cartilage has not yet been identified, but the absolute decrease in the number of chondrocytes and the imbalance of cartilage matrix synthesis and degradation in chondrocytes are known as one cause. Thus, until now, arthritis is not possible to cure the root, and the therapeutic agents developed so far are being treated to alleviate pain, suppress inflammation, and preserve possible functions.

Treatment of degenerative arthritis that is currently used clinically includes drug therapy (analgesics, steroids, nonsteroidal anti-inflammatory drugs, etc.) or cartilage protective agents (hyaluronic acid, glucosamine, chondroitin, etc.) or surgical treatment (eg, joint surgery, Proximal tibia osteotomy, joint replacement, total knee arthroplasty, etc.). However, drug therapy has only the effect of non-specifically alleviating pain or inflammatory response itself, and cartilage protector protects joints by only nourishing cartilage cells or alleviating shock.

It only plays a role. In addition, there is a side effect that can lead to osteoporosis, hypertension, diabetes, etc. due to loss of calcium when taking steroidal drugs for a long time. Therefore, in most cases, the drug is used only for the purpose of reducing pain, and permanent arthroplasty is mainly used, but there are no drugs or surgical methods that provide a fundamental therapeutic effect.

In the meantime, Houttuynia cordata Thunberg is a perennial herb belonging to the three hundred (Saururaceae) family, which has been called eoseongcho because of its 'fishy fishy' in leaves and stems. In China, Eoseongcho is widely known as a herbal medicine rather than as a medicinal herb, and it is also known as pulmonary vinegar, walnut vine and Guzai (Kim Young-young, Daesan Noncheong, Vol. 3, 188-). 196, 1995). According to the herbal tree, eoseongcho is known to be effective for fever, detoxification, diuresis, purulent and detoxification, and is applied to pertussis, bronchitis, pneumonia, tonsillitis in clinical practice, and in oriental medicine, it is used for chronic skin disease and diuresis. Yoo HT et al., Haenglim Publishing Co. , Seoul, 717-718, 1977). However, there are very few reports of arthritis of the Echo herb.

Accordingly, the inventors of the present invention, while minimizing the side effects of the conventional arthritis treatments and searching for alternatives to alleviate the pain of arthritis as much as possible, confirmed that the Echocho supercritical extract has the effect of effectively alleviating the inflammation caused by arthritis and alleviating the pain. By this, the present invention was completed.

Therefore, an object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of arthritis diseases, containing Echocho supercritical extract as an active ingredient.

Another object of the present invention to provide a food for the prevention and improvement of arthritis diseases containing the supercritical extract of Eochochocho as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of arthritis diseases containing Echocho supercritical extract as an active ingredient.

In another aspect, the present invention provides a food for the prevention and improvement of arthritis diseases, containing Echocho supercritical extract as an active ingredient.

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

Pharmaceutical composition for the prevention and treatment of arthritis diseases of the present invention is characterized in that it contains Echo supercritical extract as an active ingredient.

The Echocho supercritical extract is not limited thereto, but is preferably obtained by supercritical extraction using carbon dioxide as a solvent, and is not particularly limited as long as it is a temperature and pressure capable of creating a supercritical state. It is characterized in that the extraction at 350 ~ 450 atm, 50 ~ 70 ℃ using the carbon dioxide solvent. Meanwhile, the wax may be removed by centrifuging the supercritical extract of Eochocho supernatant at 10,000-30,000 rpm for 5-30 minutes to obtain a supernatant. Most preferably, the wax may be removed by taking the supernatant after centrifugation for 10 minutes at 18,000 rpm.

In an embodiment of the present invention, after the root, leaf or stem of Eochocho in a supercritical extraction tank after the deodorization pre-treatment process was added using a high-pressure gas pump to maintain a constant pressure and temperature at 400 atm, 60 ℃ Eochocho supercritical extract was prepared (see <Example 1>).

In addition, in one embodiment of the present invention, as a result of administering the prepared Echo supercritical extract to the macrophages of mice, it was confirmed that the anti-inflammatory activity is excellent by inhibiting the production of NO and PGE ₂ and inhibiting the activity of NF-κB ( See Example 2). In addition, the administration of the supercritical extract of the above-mentioned fish herb was administered to a carrageenan-air pouch animal model to confirm that it effectively inhibits the production of NO, PGE ₂ and TNF-α. It was found that the activity was maintained (see <Example 3>).

In addition, as a result of administering the prepared supercritical extract of Eochochochoe to Sprague-Dawley rats that induced inflammation, it was found that there was a pain suppression effect by raising the pain threshold statistically (< See Example 4).

In addition, Echo supercritical extract prepared above was induced degenerative arthritis As a result of administration to Wistar mice, it was confirmed that degenerative arthritis was alleviated (see <Example 5>).

Therefore, the Echo supercritical extract prepared above effectively inhibits NO, PGE ₂ production and NF-κB activity in vitro and also effectively inhibits NO, PGE ₂ and TNF-α production in vivo, and thus has excellent anti-inflammatory activity. Do. In addition, the Echo supercritical extract prepared above is excellent in the effect of inhibiting pain, which is the main symptom of degenerative arthritis in an actual animal model, and can be effectively used for the prevention and treatment of degenerative arthritis disease because it alleviates the degenerative arthritis. Therefore, the pharmaceutical composition of the present invention containing Echocho supercritical extract as an active ingredient is very effective for the prevention and treatment of degenerative arthritis diseases.

Meanwhile, Eochocho supercritical extract used in the present invention has a minimum lethal dose (MLD) of 5,000 mg / kg orally when administered orally (see <Experimental Example 1-1>), and does not cause a reverse mutation (Experimental Example 1). -2) Since it does not cause micronuclei in mouse bone marrow cells (see <Experimental Example 1-3>), it is considered to be a safe substance because it is not toxic, and thus the pharmaceutical composition of the present invention containing it as an active ingredient can be used safely. .

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier with Echocho supercritical extract. As used herein, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and that, when administered to a human, typically does not cause allergic or similar reactions, such as gastrointestinal disorders, dizziness, and the like.

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium, stearate, stearic acid and the like. In addition, carriers for parenteral administration may include water, suitable oils, saline, aqueous glucose and glycols, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to those described in the following references (Remington's Pharmaceutical Science, 19th Edition, Mack Publishing Company, Easton, PA, 1995).

The pharmaceutical compositions of the present invention may be prepared in various parenteral or oral administration forms according to known methods.

Preferred formulations for parenteral administration are injectable formulations such as isotonic aqueous solutions or suspension forms and formulations in the form of ointments. Injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving in saline or buffer. In addition, oral administration formulations include, but are not limited to, powders, granules, tablets, pills and capsules, and these formulations may contain, in addition to the active ingredients, diluents (e.g., lactose, dextrose, throat, mannitol, sorbitol). , Cellulose and / or glycine), suspending agents such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols. Tablets may also include defects such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethyl cellulose and / or polyvinylpyrrolidine, optionally starch, agar, alginic acid or Disintegrating or boiling mixtures such as sodium salts thereof and / or absorbents, colorants, flavors and sweeteners. The formulations may be prepared by conventional mixing, granulating or coating methods.

In addition, the route of administration of the pharmaceutical composition according to the present invention is not limited thereto, and includes oral, intravenous, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, spleen, intestinal, topical, Sublingual or rectal. Preferably the pharmaceutical composition according to the invention may be administered parenterally, such as by subcutaneous, intravenous, intramuscular, intraarticular, intramuscular, intrasternal, intradural, intralesional and intracranial injection or infusion. . For example, the pharmaceutical composition according to the present invention formulated for injection can be injected into the lower layer of the skin with a thin needle of 4-6 mm or lightly pricked the skin with a 30 gage needle. It can be administered by the method of Messo theraphy. In addition, when applied to the skin can be formulated in the same form as an ointment to be administered transdermally. As used herein, "transdermal administration" means the topical administration of the pharmaceutical composition to the skin to deliver an effective amount of the active ingredient contained in the pharmaceutical composition into the skin.

The pharmaceutical composition of the present invention may be administered to a patient in a single dose, and may be administered by a fractionated treatment protocol in which multiple doses are administered for a long time. The composition of the present invention may vary an effective amount depending on the extent of the disease, but may be administered in 0.001 to 200mg / kg, preferably 0.001 to 200mg / kg per day, but is not limited thereto. However, the concentration of Echo supercritical extract may be determined based on various factors such as the age, weight, health condition, sex, severity of the disease, diet and excretion rate, as well as the route and frequency of treatment of the drug. Can be. In view of this, one of ordinary skill in the art will be able to determine the appropriate effective dosage for the particular use as a therapeutic or prophylactic agent for degenerative arthritis.

The composition according to the present invention is not particularly limited to the formulation, route of administration and method of administration which showed the effect of the present invention.

In addition, the food composition for the prevention and improvement of arthritis diseases of the present invention is characterized in that it contains Eochocho supercritical extract as an active ingredient.

Examples of the food to which the Echochocho supercritical extract can be added include various foods, teas, vitamin complexes, health functional foods, powders, granules, tablets, capsules, and the like. It may also be added to food for the purpose of preventing and improving arthritis. At this time, the amount of the extract in the food or beverage may be added in 0.01 to 100% by weight of the total food weight, the food composition may be added in a ratio of 0.02 to 100% by weight of the total weight.

The food is not particularly limited to other ingredients except for containing the extract as an essential ingredient in the indicated ratio, and may include various flavors of natural carbohydrates or edible oils and the like as additional ingredients. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the above-mentioned flavoring agents, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. Paper is not limited to this but preferably soybean oil, olive oil, green tea seed oil, pumpkin seed oil, grape seed oil sunflower oil and canola oil and the like.

The food of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavoring agents, colorants and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and the like. Salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the food of the present invention may contain natural fruit juice and fruit flesh for the production of fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not limited thereto but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the food of the present invention.

As described above, the pharmaceutical composition or food containing the Echocho supercritical extract of the present invention as an active ingredient is excellent in anti-inflammatory activity and pain suppression effect and relieves degenerative arthritis, and thus can be effectively used for the prevention and treatment of arthritis diseases.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the present invention, the contents of the present invention is not limited to the following examples.

< Example  1>

Eoseongcho Supercritical  Preparation of Extract

10 kg of roots, leaves or stems of fish deodorant deodorized were placed in a supercritical extraction tank, and the extraction tank was sealed. The temperature of the extraction tank was raised to 60 ° C, carbon dioxide was introduced using a high pressure gas pump to raise the pressure to 400 atm. In particular, the pressure and temperature were kept constant at 400 atm and 60 ° C. while releasing the extract using a pressure control valve attached to the outlet of the extraction tank. The extract obtained from the above process was centrifuged to prepare 150 g of Echo supercritical extract (yield: 1.5%).

< Example  2>

in in vitro Eoseongcho Supercritical  Anti-inflammatory Activity of Extracts

<2-1> NO  Inhibitory activity

In order to determine whether the Echo supercritical extract prepared in Example 1 has an activity of inhibiting the production of nitric oxide, it was induced by lipopolysaccharide (LPS) using macrophages of mice (murine macrophage RAW 264.7). The amount of nitric oxide was measured by a griess reaction. Specifically, after culturing the macrophages of mice in DMEM (Dulbecco's modified Eagle's Medium), LPS and the supercritical extract of Echochochoe prepared in <Example 1> were added to each concentration and incubated for 24 hours to the nitric oxide produced The amount was measured by the griess reaction. More specifically, 100 μl of the culture solution and the same amount of the Gries reagent were added to a 96 well plate, and left for 10 minutes, after measuring the absorbance at 550 nm. The results are shown in FIG. 1.

As shown in FIG. 1, the amount of nitric oxide produced was found to decrease depending on the concentration of the supercritical extract of Eochocho supercritical extract. In particular, the IC ₅₀ value of Echo supercritical extract was measured to be 29.8 μg / ml (aminoguanidine, a positive control). IC ₅₀ values of the (aminoguanidine) is 12.5㎍ / ㎖ Im).

<2-2> PGE ₂ Inhibitory activity

To determine whether Echochocho supercritical extract has activity that inhibits PGE ₂ production, one of the inflammatory factors, incubated mouse macrophage (murine macrophage RAW 264.7) and treated with 500uM of aspirin for 3 hours to treat COX (cyclooxygenase). After inhibiting the activity of) -1, LPS (lipopolysaccharide) and the supercritical extract of Eochochochoe prepared in <Example 1> were added according to the concentration and incubated for 16 hours, and the amount of PGE ₂ generated in the culture solution was measured using EIA (Enzyme). Immunoassay Kit) and the results are shown in FIG.

As shown in FIG. 2, it was confirmed that the production amount of PGE ₂ was reduced depending on the concentration of Echocho supercritical extract. In particular, the IC ₅₀ value of Echochocho supercritical extract was measured to be 21.5 μg / ml (India, a positive control group). IC ₅₀ value of methasin (indomethacin) is 2.5 μg / ml).

<2-3> NF - κ B Inhibitory Activity

After treating macrophages (murine macrophage RAW 264.7) with NF-κB receptor constructs, LPS (lipopolysaccharide, 1 µg / ml) and Echochosperm supercritical extract prepared in Example 1 were used. After culturing for an hour, the cells were exchanged with fresh medium (200 μl) for another 48 hours. After the incubation, 100 μl of the supernatant was transferred to a 96 well plate and incubated at 65 ° C. for 5 minutes, followed by adding 100 μl of 2-fold secreted alkaline phosphatase (SEAP) buffer solution and incubating at 37 ° C. for 10 minutes. Reaction was performed by adding 20 µl of 120 mM p-nitrophenylphosphate (p-nitrophenylphosphate). After the reaction, the absorbance was measured at 405 nm using a microplate reader and the results are shown in FIG. 3.

As shown in FIG. 3, the supercritical extract of Eochochoea was found to inhibit NF-κB, and the IC ₅₀ value of Echochori supercritical extract was measured to be 29.0 μg / ml.

Eochocho supercritical extract of the present invention as described above in The anti-inflammatory activity is excellent by inhibiting the production of nitric oxide and PGE ₂ and inhibiting the activity of NF-κB in vitro . Therefore, it may be useful as a pharmaceutical composition for the prevention and treatment of arthritis, especially degenerative arthritis disease.

< Example  3>

in vivo Eoseongcho Supercritical  Anti-inflammatory Activity of Extracts

<3-1> NO  Inhibitory activity

First in Carrageenan-air pouch animal model was prepared to determine whether Echochocho supercritical extract has nitric oxide inhibitory activity in vivo .

Specifically, the hair of an animal was depilated with an electric razor, and then lightly anesthetized with ether, and 10 ml of mice and 20 ml of sterile air were injected subcutaneously. After 2 and 5 days, 5 ml of mice and 10 ml of sterile air were additionally injected into rats, respectively. Twenty-four hours after injection, test substances (20, 65 or 200 mg / kg) were orally administered and 30 minutes later, 1 ml of carrageenan solution dissolved in 1% in sterile saline was injected into mice and 2 ml of rats in an air swell. . Positive controls dexamethasone (2 mg / kg) and indomethacin (2 mg / kg) were injected intraperitoneally at the same time as the test substance. After 6 hours, 1 ml of sterile saline was injected into the rat and 2 ml of sterile saline into the rat, and an excision was collected by incision.

The recovered exudate was measured through a known enzyme immunoassay (EIA) and a grease reaction (Griess reaction) to measure the amount of nitric oxide that is an inflammatory reaction product. Inhibition rate was calculated by the following Equation 1 using the amount produced and the results are shown in Table 1 below.

Inhibition rate = {(carrageenan-non-administered group)-(experiment group-non-administered group)} / (carrageenan-non-administered group) * 100

Inhibitory Activity of Nitric Oxide Production by Echochorea Supercritical Extract (Mean ± S.E.M.) Dosage substance Nitrogen oxide production amount (pg / mL) % Inhibition Non-administered group 0.391 ± 0.211 - Carrageenan 0.774 ± 0.320 - Carrageenan + dexamethasone (2 mg / kg) 0.054 ± 0.067 188.0 Carrageenan + indomethacin (2mg / kg) 0.663 ± 0.580 29.0 Carrageenan + Echoseong supercritical extract (20mg / kg) 0.384 ± 0.378 101.8 Carrageenan + Echoseong supercritical extract (65mg / kg) 0.412 ± 0.356 94.5 Carrageenan + Echoseong supercritical extract (200mg / kg) 0.403 ± 0.533 96.9

As shown in Table 1, the production amount of nitric oxide was confirmed to be reduced by administering the superlative supercritical extract, it can be seen that the superlative supercritical extract of the present invention has the inhibitory activity of nitric oxide production even in vivo .

<3-2> PGE ₂ Inhibitory activity

The exudates recovered from the Carrageenan-air pouch animal model prepared in Example 3-1 were measured for PGE ₂ production through known enzyme immunoassay (EIA) and Greases reaction. . Inhibition rate was calculated through Equation 1 using the amount produced, and the results are shown in Table 2 below.

Inhibitory Activity of PGE ₂ Production by Supercritical Extracts from Estuary (Mean ± SEM) Dosage substance Production amount of PGE ₂ (pg / ml) % Inhibition Non-administered group 1,328.1 ± 125.2 - Carrageenan 1,439.5 ± 18.4 - Carrageenan + dexamethasone 1,411.3 ± 67.7 25.3 Carrageenan + Indomethacin 1,222.3 ± 31.5 195.0 Carrageenan + Echoseong supercritical extract (20mg / kg) 963.8 ± 134.3 427.0 Carrageenan + Echoseong supercritical extract (65mg / kg) 1,149.6 ± 147.4 260.2 Carrageenan + Echoseong supercritical extract (200mg / kg) 1,300.7 ± 117.8 124.6

As shown in Table 2, the production amount of PGE ₂ was confirmed to be reduced by administering the Echo supercritical extract, in particular the Echo supercritical extract of the present invention in In vivo , it was found that PGE ₂ production inhibitory activity.

<3-3> TNF -α inhibitory activity

The amount of TNF-α was measured using a known Enzyme-linked immunosorbent assay (ELISA) for the exudates recovered from the Carrageenan-air pouch animal model prepared in <Example 3-1>. Inhibition rate was calculated through Equation 1 using the amount produced, and the results are shown in Table 3 below.

TNF-α Inhibitory Activity (Mean ± S.E.M.) Dosage substance Production amount of TNF-α (pg / mL) % Inhibition Non-administered group 5.0 ± 0.1 - Carrageenan 181.7 ± 142.8 - Carrageenan + dexamethasone 14.1 ± 16.5 94.9 Carrageenan + Indomethacin 23.6 ± 22.2 89.5 Carrageenan + Echoseong supercritical extract (20mg / kg) 95.8 ± 45.1 48.6 Carrageenan + Echoseong supercritical extract (65mg / kg) 56.7 ± 43.3 70.7 Carrageenan + Echoseong supercritical extract (200mg / kg) 79.1 ± 57.4 58.1

As shown in Table 3 above, Echo supercritical extract of the present invention can be seen to exhibit an excellent inflammation inhibitory effect by inhibiting TNF-α production. Therefore, Echocho supercritical extract of the present invention is considered to be useful as a pharmaceutical composition for the prevention and treatment of arthritis, especially degenerative arthritis disease.

< Example  4>

Eoseongcho Supercritical  Inhibitory Effect of Extracts

In order to confirm the pain-inhibitory effect of Echocho supercritical extract of the present invention, according to Randall, LO and Selitto, JJ, Arch . Int . Pharmacodyn . Ther . , 111, 4.9-19, 1971 The same experiment was performed.

First, overnight Sprague-Dawley rats were fasted overnight to perform the experiment in inflamed rats, and then 0.1 ml of saline suspension containing 20% brewers yeast was added to each rat's hind paw. Inflammation was induced by subcutaneous injection, and then separated into five groups of eight animals. In this case, the G1 group was used as an excipient control group, and the G2 to G4 groups were administered Echochochocritic extract prepared in Example 1 above. That is, 20 mg / kg in the G2 group, 65 mg / kg in the G3 group and 200 mg / kg in the G4 group. Meanwhile, G5 group was administered 10 mg / kg of indomethacin (indomethacin). After 0, 0.5, 1, 2, 3, 4 hours, the pain threshold (g) was measured with an analyte. The pain threshold is gradually applied to the rat's hind foot at a rate of 16 g / sec. The pain response is when the rat tries to pull out the foot or twists the body due to pain and analyzes the result statistically by Student's t-test. 4 is described.

Inhibitory Effect of Pain in Inflamed Rats (Mean ± S.E.M.) group 0 hours 0.5 hour 1 hours 2 hours 3 hours 4 hours G1 109.0 ± 6.0 82.5 ± 8.1 84.5 ± 7.0 62.5 ± 2.4 60.8 ± 5.1 52.3 ± 4.5 G2 116.5 ± 9.4 106.3 ± 12.6 97.5 ± 10.1 82.5 ± 7.2 ^* 72.8 ± 11.9 59.8 ± 6.4 G3 117.3 ± 6.1 112.5 ± 11.2 ^* 129.3 ± 16.1 ^* 94.0 ± 7.9 ^** 74.8 ± 8.1 65.3 ± 9.0 G4 113.5 ± 3.6 113.0 ± 10.2 ^* 132.3 ± 6.0 ^** 92.3 ± 10.1 ^* 66.3 ± 8.0 62.5 ± 7.0 G5 103.8 ± 5.5 147.0 ± 16.5 ^** 150.5 ± 7.8 ^** 125.3 ± 15.9 ^** 73.3 ± 12.6 69.5 ± 7.9

* Significant difference from excipient control at P <0.05.

** Significant difference from excipient control at P <0.01.

As shown in Table 4 and Figure 4, Eochocho supercritical extract was found to have a pain inhibitory effect by increasing the pain threshold statistically significantly in the rats induced inflammation.

< Example  5>

Eoseongcho Supercritical  Effect of Extracts on the Prevention and Treatment of Degenerative Arthritis

Wistar family mice were randomly divided into 5 groups of 10 rats. The test substance was administered for 6 weeks after the separation, and then degenerative arthritis was induced at the 7th week. Specifically, 3 mg / joint of MIA (mono iodoacetate) was injected once at a volume of 50 μl in the joint cavity. Thereafter, the test substance is administered up to 8 parking days, followed by fasting on the day before necropsy, body weight is measured on the day of necropsy, the joint tissue is fixed in a 10% neutral buffered formalin solution, and histopathological examination is performed. Histopathological examination was performed to read the efficacy on the effect on arthritis based on the indexes listed in Table 5 and Table 6. The arthritis index was calculated by dividing into four items in the pathologies of the osteoarthritis-induced femur and tibia, and compared with the positive control group. The relative evaluation was performed based on the following criteria and the results are shown in Table 7.

Significance test by Kruskal-Wall's H-test.

* Significant at P <0.05 compared to G1.

** Significant at P <0.01 compared to G1.

 At this time, G1 group was administered only soybean oil (Soybean oil) as an excipient control group, G2 to G4 group was orally administered the Echo supercritical extract prepared in Example 1. Specifically, 20 mg / kg in the G2 group, 65 mg / kg in the G3 group and 200 mg / kg in the G4 group were administered daily. Meanwhile, the G5 group orally administered 5 mg / kg of the positive control substance naproxen daily. In order to confirm whether degenerative arthritis was alleviated around the contact area of chondrocytes 8 weeks after the administration, the area was observed under a microscope (X200) by H & E staining and the results are shown in FIGS. 5 to 10.

 As shown in Table 7, the control group (G1) that caused only arthritis without the treatment of the material showed a noticeable damage in the joint area and the overall structure of the joint. On the other hand, in the test substance-administered group, the dose-related visual improvement effect began to appear from G3. In the high-dose group, G4, statistically significant improvement was found in the contact area of chondrocytes (p <0.01). In the G5 group, the statistically significant improvement was observed in the joint structure (p <0.05). Overall joint damage was 11.99 in G1 and 6.25 and 7.84 in G4 and G5, respectively. The higher mean of G5 damage than G4 was due to severe joint damage in some subjects (animal No. 44). Therefore, the visible improvement in arthritis was evaluated in G4 and G5.

In addition, as shown in FIG. 5, in the normal mouse without degenerative arthritis, two to three layers of cells with smooth surface areas and flat nuclei were observed in the surface layer. In addition, in the transitional zone (A), rounded chondrocytes were observed and proliferative chondrocytes were observed. It can be seen that the hardened region B and the transitional / radial zone are separated.

However, as shown in FIG. 6, in the G1 group in which degenerative arthritis is induced, the transition and radiation regions are lost in the entire structure of the joint and the area where the joint is in contact with the normal mouse of FIG. 5. An inflammatory output in the inner layer, pannus (arrow pointing area), was observed on the surface.

On the other hand, in the G2 group treated with Echocho supercritical extract, as shown in FIG. 7, the cartilage surface was abnormal, the cells were lost in the tangential region, and the chondrocytes and the hardened region were disappeared (black arrow portion). ). It was also found that pannus formed and increased along the cartilage surface (white arrow area).

In addition, as shown in Figure 8, in the case of G3 group treated with Eochocho supercritical extract, the cartilage surface was normal and the cells of the tangential region were increased, and the cells of the transition and radiation zones (arrow part) showed high proliferation. You can see that disappeared.

In addition, as shown in Figure 9, in the G4 group treated with Echocho supercritical extract, the tangential region was normal, the chondrocytes increased significantly in the transition and radiation zones (arrows) and the pannus disappeared.

On the other hand, in the G5 group treated with naproxen as a positive control material as shown in Figure 10 it can be seen that the chondrocytes (arrow portion) is normal in the tangential region and transition / radiation region.

Therefore, Echocho supercritical extract of the present invention was found to be effective in treating degenerative arthritis even in a real animal model.

Experimental Example 1

Toxicity Test of Eochocho Supercritical Extract

<1-1> Toxicity test by oral administration

In order to determine the acute toxicity of Echochocho supercritical extract of the present invention, an acute toxicity test was carried out as follows.

Acute toxicity experiments were conducted using Sprague-Dawley rats. Five female and male animals per group were orally administered with 0, 1,000, 2,000 or 5,000 mg / kg, respectively, of the Echo-supercritical extract prepared in <Example 1>. Echocho supercritical extract of the present invention does not show a change in toxicity even up to 5,000 mg / kg in rats, the minimum lethal dose (MLD) when administered orally was determined to be more than 5,000 mg / kg safe material.

<1-2> Return mutation test

Salmonella typhimurium strains (TA100, TA1535) and E. coli WP2 uvrA were used to determine whether Echochocho supercritical extracts induced base-pair substitution type mutations. TAshift and TA1537 strains were used to determine whether they cause the -shift type mutation.

To determine the appropriate dosage concentration, the highest dose was determined at 10,000 μg / plate, which showed a decrease in colony when no metabolic activity of the TA1537 and E. coli strains was observed, but no increase or decrease in the number of colonies was observed in other strains. 185, 556, 1667, 5000 and 10000 μg / plate were administered for each strain.

Direct plate incorporation was performed to administer the Echochocho supercritical extract. Specifically, dispensing 2 ml of top agar into a sterile tube, and then mixing the supercritical extract of Eochocho, 0.5 ml of S-9 mix, and 0.1 ml of the culture medium to the top agar at 37 ° C. After approximately 48 hours of incubation, the mutant colonies were counted. At this time, 0.1 ml of excipient was used as a negative control group, and DMSO solution was used as a positive control group.

As a result, the antimicrobial activity was reduced by 0.5 times compared to the negative control at the highest concentration when the metabolism activity of TA1535 was not applied and the concentration of 5,000ug / plate when TA1537 was applied and not applied. ) Thinning or disappearing) was not observed. Other strains did not show an increase or decrease in the number of colonies compared to the negative control. On the other hand, the number of colonies increased in all positive controls compared to negative controls.

Therefore, Eochocho supercritical extract of the present invention was determined to be a safe substance because it does not cause a back mutation in the test strain used under the present test conditions.

<1-3> oral administration Micronucleus  exam

Thirty seven-week-old ICR mouse males were divided into five groups and subjected to micronucleus induction test by Echocho supercritical extract. At this time, the following dose was determined based on preliminary experiments.

Oral Micronucleus Test Method Administration group Animal count (horses) Dose (mg / kg / day) Number of doses Dosage amount (ml / kg / day) Route of administration MMPCE / 2000 PCE (mean ± S.D.) PCE / (PCE + NCE) (mean ± S.D.) Negative Control G1 6 0 2 10 oral- 0.83 ± 0.75 0.37 ± 0.01 Administration group G2 6 1,250 2 10 oral- 0.83 ± 0.75 0.39 ± 0.02 * Administration group G3 6 2,500 2 10 oral- 0.67 ± 0.82 0.39 ± 0.01 Administration group G4 6 5,000 2 10 oral- 1.17 ± 1.47 0.38 ± 0.01 Positive control group G5 ^a) 6 70 One 10 Abdominal cavity 62.17 ± 15.09 ** 0.24 ± 0.01 **

a) Cyclophosphamide H ₂ O

Significance test by Kruskal-Wall's H-test.

* Significant at P <0.05 compared to G1.

** Significant at P <0.01 compared to G1.

Bone marrow cells obtained according to the known Schmid (1975) method were plated on slide glass, dried sufficiently at room temperature, and the cells were fixed with methanol. The fixed and dried samples were subjected to 3 minutes in May-Grunwald dye solution, 2 minutes in May-Grunwald dye solution and distilled water mixed 1: 1, and in Giemsa stain solution (5% in PBS, pH6.8). Stained for 10 minutes and examined at 1,000 times magnification. To calculate the frequency of micronuclei, we counted the number of micronucleated polychromatic erythrocytes (MPNs) in micronucleated polychromatic erythrocytes (MPNs) out of 2,000 PCEs (polychromatic erythrocyte polyinflammatory erythrocytes) per animal. Micronucleus frequency was expressed as the mean ± S.D. Of the number of MNPCE observed at 2,000 PCE per individual. More than 500 PCE and NCE (normochromatic erythrocytes) were counted with or without micronucleus to calculate the PCE / (PCE + NCE) ratio, ie the percentage of polyinflammatory red blood cells in total red blood cells, as an indicator of cytotoxicity. .

As a result of counting micronuclei of 2,000 PCEs per subject within the dose range applied to this test, there was no statistically significant change in all administration groups administered test substance compared to the negative control group, and it was an indicator of cytotoxicity. Phosphorus PCE / (PCE + NCE) ratio was above 0.39 in all groups treated with test substance and increased significantly in 1,250mg / kg / day dose group (P <0.05), which was not significant in terms of genotoxicity. It was.

Therefore, Echocho supercritical extract of the present invention did not cause micronuclei in mouse bone marrow cells under this test condition and was determined to be a safe substance.

Production Example 1 Preparation of Health Food

Eochocho Supercritical Extract ...... 1000 mg

Vitamin mixture proper amount

Vitamin A Acetate ............... 70 μg

Vitamin E .................................. 1.0 mg

Vitamin B1 ................................. 0.13 mg

Vitamin B2 ................................. 0.15 mg

Vitamin B6 ................................. 0.5 mg

Vitamin B12 ......... 0.2 μg

Vitamin C ......................................... 10 mg

Biotin .......................... 10 μg

Nicotinic Acid Amide ...................................... 1.7 mg

Folic acid ......................................... 50 μg

Calcium Pantothenate ......................................... 0.5 mg

Mineral mixture

Ferrous Sulfate ......... 1.75 mg

Zinc Oxide ........................ 0.82 mg

Magnesium Carbonate ............... 25.3 mg

Potassium monophosphate ......................................... 15 mg

Dicalcium Phosphate Dioxide ..................... 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.

Production Example 2 Preparation of Healthy Drinks

Eochocho Supercritical Extract ............... 1000 mg

Citric Acid ......................................... 1000 mg

Oligosaccharide ........................... 100 g

Plum concentrate ........................... 2 g

Taurine ............... 1 g

Purified water was added and the above ingredients were mixed according to the general method for preparing a healthy beverage, and then stirred and heated at 85 ° C. for about 1 hour, and then the resulting solution was collected by filtration in a sterilized 2 L container and sealed and sterilized. It is then refrigerated and then used to prepare a healthy beverage composition of the present invention. Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

1 is a graph showing the measurement of nitric oxide production inhibitory activity of Echocho supercritical extract.

Figure 2 is a graph showing the measurement of PGE ₂ production inhibitory activity of Eochocho supercritical extract.

Figure 3 is a graph showing the measurement of NF-κB inhibitory activity of Eochocho supercritical extract.

Figure 4 is a graph showing the measurement of the pain inhibitory effect in the inflammation-induced rats Eochocho supercritical extract.

* Significant difference from excipient control at P <0.05.

** Significant difference from excipient control at P <0.01.

5 is a microscopic view of cartilage tissue of normal mice without degenerative arthritis (A: transition region of chondrocytes, B: hardened region).

6 is a photograph of the cartilage tissue of a mouse in which degenerative arthritis is induced through a microscope (arrow indicating portion: pannus).

FIG. 7 is a photograph of cartilage tissue observed through a microscope after administration of Eochocho supercritical extract (20 mg / kg) to mice with degenerative arthritis.

FIG. 8 is a photograph of cartilage tissue observed through a microscope after administration of Echo supercritical extract (65 mg / kg) to mice with degenerative arthritis.

Figure 9 is a photograph of the cartilage tissue observed after the administration of the superlative supercritical extract Eochocho supercritical extract (200mg / kg) to mice with degenerative arthritis through a microscope.

FIG. 10 is a photograph of cartilage tissue observed under a microscope after administration of a positive control substance naproxen (5 mg / kg) to mice with degenerative arthritis.

Claims (7)

Pharmaceutical composition for the prevention and treatment of arthritis diseases containing Echocho supercritical extract as an active ingredient. The pharmaceutical composition of claim 1, wherein the arthritis is degenerative arthritis. The pharmaceutical composition according to claim 1, wherein the supercritical extract is obtained by supercritical extraction using carbon dioxide as a solvent. According to claim 2, wherein the supercritical extraction is a pharmaceutical composition, characterized in that the extraction at 300 ~ 500 atm, 30 ~ 80 ℃. According to claim 4, wherein the supercritical extraction is a pharmaceutical composition, characterized in that the extraction at 350 ~ 450 atm, 50 ~ 70 ℃. Food for the prevention and improvement of arthritis diseases, containing Echocho supercritical extract as an active ingredient. 7. The food of claim 6, wherein the arthritis is degenerative arthritis.

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