KR101023487B1 - Arthritis prevention or treatment composition comprising a mixed herbal extract of Schisandra chinensis, golden and thawed skin as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of arthritis comprising a mixed herbal extract of Schisandra chinensis, golden and thawed skin as an active ingredient. The mixed herbal extracts of Schisandra chinensis, golden and thawed blood contained in the composition of the present invention inhibit the synthesis and degradation of extracellular matrix components of chondrocytes including hyaluronic acid in the joints commonly occurring in osteoarthritis patients and are the main symptoms of arthritis diseases. Since the edema and pain can be alleviated, the composition of the present invention comprising such mixed herbal extract as an active ingredient can be usefully used for preventing or treating arthritis.

Description

Composition for Prevention or Treatment of Arthritis Comprising the Complex Herb Extract of Schisandra Chinensis Bail, Scutellaria Baicalensis and Kalopanax Pictus Nakai}

The present invention relates to a composition for preventing or treating arthritis, comprising a mixed herbal extract of Schisandra chinensis Bail, Scutellaria baicalensis, and Kalopanax pictus Nakai, as an active ingredient, and a method for preparing the same. will be.

Arthritis is a representative of cartilage-related diseases, and is classified into rheumatoid arthritis and degenerative arthritis (osteoarthritis), and knee joint cartilage is often found in traffic accidents or excessive exercise. At present, there are about one million arthritis patients in Korea, and women are more than twice as many as men, especially menopausal women (Belmonte-S. MA et al; Arthritis Rheum, 30, pp 1214-). 1225, 1987).

Rheumatoid arthritis is a type of autoimmune disease, and it occurs in women of 30 to 50 years at a ratio of 1: 4. Symptoms include symmetrical inflammation of the joints of fingers, elbows, knees, etc. It is severe at dawn, weakness and mild fever. Arthritis in acute joint rheumatism disappears within a few days to weeks, whereas rheumatoid arthritis lasts for months and years, resulting in joint deformation, dislocations, muscle tendon buildup and stiffness. Causing severe disability (Feldmannn, et al; Cell, 85: pp 307-310, 1996).

Degenerative arthritis is a disease in which local degenerative changes occur as the articular cartilage wears down, 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 or overweight, and appear more and more severely in women as they age (Oliver, JE et al; Scand J Rheumatol, 35, pp 169-174, 2006). The initial symptom is pain in one or two joints with stiffness, and if prolonged, the joint will be deformed. 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. Therefore, degenerative changes in articular cartilage decrease cartilage strength and ability as a cushion due to the breakdown of cartilage matrix (Loeser, RF et al; Arthritis Rheum, 54, pp 1357-1360, 2006).

Currently used clinically treated degenerative arthritis can be treated with medications (painkillers, steroids, nonsteroidal anti-inflammatory drugs, etc.) or cartilage protectants (hyaluronic acid, glucosamine, chondroitin, etc.) or surgical treatment (arthroscopy, proximal tibia). Osteotomy, joint replacement, total knee arthroplasty, etc. (Zhang, B et al; Eur J Pharmacol, 530, pp 166-171, 2006).

Hyaluronic acid, which is used as a cartilage protective agent, is a polymer compound in which N-acetylglucosamine and glucuronic acid are alternately bonded in a chain shape and has a molecular weight of about 20 to 400,000, and is known as a major mucopolysaccharide together with chondroitin sulfate. It exists in the vitreous body of the eye and the umbilical cord, and is viscous and is important for preventing the invasion of bacteria and the infiltration of poison. This hyaluronic acid is similar to the pectin of plants and is hydrolyzed by hyaluronidase. Hyaluronic acid having the above characteristics is used as a therapeutic agent for arthritis because it is combined with a large amount of water and becomes a gel state and is involved in lubrication of joints and skin flexibility, and is directly degenerated by injection directly into the main component of cartilage synovial fluid. It is used as a substitute for depleted cartilage synovial fluid.

However, drug therapy has only the effect of non-specifically relieving pain or inflammatory response itself, and cartilage protection agent only serves to protect joints by nourishing chondrocytes or alleviating shock. In addition, steroidal agents have side effects that can lead to osteoporosis, high blood pressure, diabetes, etc., due to loss of calcium when taken 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. Although much research and efforts have been made for the treatment of degenerative arthritis in the past decades, the pathological cause and the underlying treatment are not yet developed. Recently, the treatment of arthritis through herbal and nutritional supplements has been developed, but the therapeutic effect and mechanism of action are not known (Gaby, AR et al; Antern Med Rev, 4, pp 392-402, 1999). Therefore, these arthritis diseases are not only difficult to effectively control the disease with the simple anti-inflammatory drugs, the existing simple symptomatic therapy, but also cause a lot of difficulties in the treatment of the elderly, which is the majority of patients in reality due to the side effects of long-term administration of the drug. Therefore, in order to treat arthritis, since it is necessary to take a medicament for a long time, development of a medicament with few side effects is required.

Therefore, the inventors of the present invention while conducting research on the inhibition of hyaluronidase activity and physiological activities related to inflammation in the herbal extracts, the mixed herbal extracts of Schisandra chinensis, golden and thawed blood are not toxic and inflammatory in articular chondrocytes and tissues. The present invention is completed by inhibiting the synthesis of substance free substance and substrate destroying substance and inhibiting pathological progression such as pain and swelling of arthritis, thereby revealing that it can be usefully used as a therapeutic agent or preventive agent or health functional food without side effects. It was.

The present invention provides a composition for the prevention or treatment of arthritis, containing the mixed herbal extract of Schisandra chinensis, golden and thawed skin as an active ingredient.

In addition, the present invention also provides a health functional food for preventing or treating arthritis, which contains a mixed herbal extract of Schisandra chinensis, golden and thawed skin as an active ingredient.

In addition,

(a) extracting a mixed herbal medicine mixed with Schisandra chinensis, golden and thawed blood using alcohol;

(b) concentrating the alcohol extract under reduced pressure and freeze drying to obtain a powder; And

(c) The powder obtained in step (b) is first fractionated using chloroform and the residue is fractionated using ethyl acetate to provide a mixed herbal medicine extract manufacturing method comprising the step of obtaining a final mixed herbal medicine extract.

The mixed herbal extracts of Schisandra chinensis, golden and thawed blood contained in the composition of the present invention inhibit the synthesis and degradation of extracellular matrix components of chondrocytes including hyaluronic acid in the joints commonly occurring in osteoarthritis patients and are the main symptoms of arthritis diseases. In order to relieve edema and pain, the composition of the present invention comprising such a mixed herbal extract as an active ingredient can be usefully used for the prevention or treatment of arthritis.

Figure 1 is a photograph showing the inhibitory activity against hyaluronidase (hyaluronidase) of the mixed herbal extract YD101 and the extract alone (SCEA, SBEA, KPEA) of the present invention.
Figure 2 is a photograph showing the inhibitory activity against the cartilage matrix component degrading enzyme collagenase of each of the mixed herbal extract YD101 and the extract alone (SCEA, SBEA, KPEA) of the present invention.
Figure 3 is a graph showing the inhibitory activity against cartilage matrix component enzyme elastase (elastase) of the mixed herbal medicine extract of the present invention YD101.
Figure 4 is a graph showing the degradation inhibitory effect of the cartilage matrix component of the cartilage matrix in the cartilage tissue culture medium of the mixed herbal extract YD101 and the extract alone (SCEA, SBEA, KPEA) of the present invention.
Figure 5 is a graph showing the effect of inhibiting the production of inflammatory substances, prostaglandin E2 (PGE2) in chondrocytes and tissues of the mixed herbal extract YD101 and the extract alone (SCEA, SBEA, KPEA) of the present invention.
Figure 6 is a graph showing the inhibitory effect on the mouse ear edema of the mixed herbal extract YD101 and the extract alone (SCEA, SBEA, KPEA) of the present invention.
Figure 7 is a graph showing the results of comparing the anti-arthritis efficacy in the mouse CIA model of the mixed herbal extracts of the present invention YD101 and comparative mixture extract SSEA (Schisandra + Golden), SPEA (Schisandra + thaw).
Figure 8 is a graph showing the results of comparing the anti-arthritis efficacy of YD101, the mixed herbal medicine extract of the present invention with the control drug celecoxib (100mg / ㎏).
Figure 9 is a photograph of the left and right Fuji showing arthritis-induced symptoms of the mixed herbal extract YD101 and the control drug celecoxib of the present invention.
FIG. 10 shows the inflammation, bone resorption, bone resorption, and bone graft formation of the foot tissue isolated from the individual group of the YD101 administration group, 0.5% CMC, which is the mixed herbal medicine extract of the present invention, and the celecoxib administration group. This is a graph showing the results of qualitative analysis based on the degree of osteophyte formation) and cartilage damage.

The present invention provides a composition for preventing or treating arthritis, comprising a mixed herbal extract of Schisandra chinensis, golden and thawed skin as an active ingredient.

In one embodiment of the present invention, the arthritis may be degenerative osteoarthritis or rheumatoid arthritis.

Hereinafter, the mixed herbal medicine extract of Schisandra chinensis, golden and thawed blood is simply referred to as 'mixed herbal medicine extract'.

First, the Schizandra chinensis, golden and thawed blood constituting the mixed herbal medicine extract will be described in detail.

The scientific name of `` Omiza '' is Schisandra chinensis Bail, deciduous broad-leaved vine, leaves alternate, ovate or obovate, with sharply pointed, jagged, somewhat hairy on back vein. Flower is reddish white and blooms in June ~ July. Fruits are used for food and hang on the ends of branches. They ripen red in August-September. Fruits are called Schizandrae fructus and Schizandrae fructus. 200 ~ 1,600m above sea level Jirisan, Jeonbuk, and Gangwon-do are the main production areas, and are wild throughout the country except Chungnam and Chungbuk, and geographically distributed in Japan, Sakhalin, Manchuria, and China. Fruits include Cizandrol, Schizandrin A, B, C (Ikeya, Y et al; Chem Pharm Bull, 35, p39-74, 1988), angeloylgomisin O, P, epigomisin, pregomoisin, deoxyschizandrin and the like (Taguchi, H et al; Chem Pharm Bull, 23, p 32-96, 1988). Schisandra chinensis has long been used in herbal medicine as a raw material of medicinal herbs and has long-standing effects such as astringent, nourishment, tonic, ginhae medicine, haejudok, thirsty, astringent shovel, ripe ginjin and bosin salt. As a result, human stability has been confirmed according to the intake of the body. Schisandra chinensis extract stimulates the central nervous system, improves tonic action, intellect and labor capacity, and affects glucose metabolism to promote catabolism of hepatic glycogen, increasing glycogen content, promoting fatigue recovery, controlling blood pressure, and secreting gastric juice. It is used to regulate blood pressure, arteriosclerosis, diabetes and hepatitis. It is also used in cosmetics because it has anti-oxidant effects, anti-bacterial detoxification, eczema and soothing inflammation, anti-aging, antibacterial and whitening effects.

The scientific name of 'golden' is Scutellaria It is baicalensis and grows in the grassland of mountain. It is 20 ~ 60cm high. Several generations come out, grow up in hair, and have branches. The leaves are opposite each other, lanceolate, with flat edges. Flowers bloom in July-August, turn purple, and run toward the side with gunshot flowers. Corollas curled at the bottom, stand straight, tubular, 1.5-2.5 cm long, and have a lip shape. Calyx is bell shaped. Two of the four surgeries are long, one pistil, and the ovary is deeply divided into four. Fruits are fruiting in September, rounded in calyx. The cone is conical and the flesh is yellow. Gold contains significant amounts of pre-non-ring flavonoids including baicalein, baicalin, wogonin and baicalenoside. Gold is used as an antipyretic, diuretic, branch, edema and anti-inflammatory agent in roots.

The scientific name of Haedongpi is Kalopanax pictus Nakai is an old tree belonging to the Araliaceae family, and the oak tree ( Kalopanax) pictus Nakai) and its related plant, the persimmon tree ( Kalopanax) pictus Nakai var. chinensis Nakai), is the bark of the fur kalopanax (Kalopanax pictus Nakai var. magnificus Nakai ), thin leaves kalopanax (Kalopanax pictus Nakai var. maximowiczii Nakai ). The components of thawed skin include 13 to 30% of tannin, glucose, kalotoxin, kalosaponin, liriodendrin, hederagenin, arabinose and benzoic acid. (Benzoic acid), amino acids (Amino acid), di-mannitol and polyacetylene-based compounds are known, the main components such as saponin, phenolic glycosides are known. Thawing blood is widely used in folk medicine such as neuralgia, arthritis, low back pain, spear and diarrhea, toothache, diabetes, tonic, and is currently sold as a folk medicine for rheumatoid and degenerative arthritis (Ono, K et al; Chem Pharm). Bull, 37, p 1810, 1989).

Mixed herbal extracts as an active ingredient of the composition of the present invention may be prepared according to conventional methods known in the art.

For example, it may be an extract prepared by mixing dried Schisandra chinensis, golden and thawed skin, and then applying hot water extraction or solvent extraction.

Meanwhile, the mixed herbal medicine extract included in the composition of the present invention may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze drying or spray drying of the primary extract extracted by the hydrothermal extraction or solvent extraction. The primary extract may be further purified using various chromatography such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, and the like. You can also get

Therefore, the mixed herbal medicine extract according to the present invention is a concept including all the extracts, fractions and purified products obtained in each step of extraction, fraction or purification, their dilutions, concentrates or dried products.

The composition for preventing or treating arthritis according to the present invention may be a pharmaceutical composition or a food composition.

A pharmaceutical composition comprising the mixed herbal medicine extract of the present invention as an active ingredient may be prepared using a pharmaceutically acceptable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant may include excipients, disintegrants, sweeteners, Binders, coatings, swelling agents, lubricants, lubricants, flavoring agents, and the like can be used.

The pharmaceutical composition may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredient for administration.

Formulation forms of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, nontoxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include but are not limited to natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, corn sweeteners, acacia, trackercance or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be formulated according to each disease or component, as appropriate in the art.

The food composition comprising the mixed herbal medicine extract of the present invention as an active ingredient may be formulated in the same manner as the pharmaceutical composition, used as a functional food, or added to various foods. Foods to which the composition of the present invention may be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice creams, alcoholic beverages, vitamin complexes, and health supplements. There is this.

At this time, the amount of the mixed herbal medicine extract added to the food is preferably 0.01 to 30 parts by weight based on the total food weight. In particular, when food is a beverage, it may be added in a ratio of 0.001 to 3g, preferably 0.1 to 3g based on the total beverage composition 100. If the amount is less than 0.001g can not expect a clear arthritis prevention or treatment effect, if the amount is added more than 3g the bitter taste of the mixed herbal medicine extract is strong to interfere with the taste of the food itself.

The present invention also provides a health functional food for preventing or treating arthritis comprising the food composition.

The functional food for preventing or treating arthritis may be beverages, meat, chocolate, food, confectionary, pizza, ramen, other noodles, gum, ice cream, alcoholic beverages, vitamin complexes or health supplements.

The present invention also provides the use of a composition comprising the above-mentioned herbal extract as an active ingredient for the prevention of arthritis, the treatment of a medicament or the manufacture of food. The composition of the present invention containing the above-mentioned mixed herbal medicine extract as an active ingredient may be used for the manufacture of a medicament or food for the prevention or treatment of diseases related to arthritis.

The present invention also provides a method for preventing or treating arthritis, comprising administering to a mammal a therapeutically effective amount of a mixed herbal extract.

As used herein, the term “mammal” refers to a mammal that is the subject of treatment, observation or experiment, preferably human.

As used herein, the term 'therapeutically effective amount' refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as thought by a researcher, veterinarian, doctor or other clinician, Amounts that induce alleviation of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dosages and frequency of administrations for the active ingredients of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by one skilled in the art and includes the type of disease, the severity of the disease, the amount of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, general health of the patient. , Sex and diet, time of administration, route of administration and rate of composition, duration of treatment, drugs used concurrently can be adjusted according to a variety of factors. In the treatment method of the present invention, in the case of an adult, the mixed herbal medicine extract of the present invention may be administered at a dose of 1 / kg to 500 / kg once or several times a day, and preferably 150 / kg to Administration at a dose of 250 / kg is recommended.

In the treatment method of the present invention, the mixed herbal extract of the present invention may be administered in a conventional manner through oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes. have.

The mixed herbal medicine extract of the present invention is a drug that can be used with confidence even when taken for a long time for the purpose of prevention because there is little toxicity and side effects.

The invention also

(a) extracting a mixed herbal medicine mixed with Schisandra chinensis, golden and thawed blood using alcohol;

(b) concentrating the alcohol extract under reduced pressure and freeze drying to obtain a powder; And

(c) The powder obtained in step (b) is first fractionated using chloroform and the residue is fractionated using ethyl acetate to provide a mixed herbal medicine extract manufacturing method comprising the step of obtaining a final mixed herbal medicine extract.

In the step (a), the weight ratio of Schisandra chinensis: Golden: thawed blood is preferably 2: 1: 1, and it is preferable to extract 2 to 4 times by adding 5 to 15 alcohols per 1 g of the mixed herbal medicines.

The method may further include the step of filtering the alcohol extract extracted in the step (a) using filter paper before the decompression concentration in step (b).

Advantages and features of the present invention and methods for achieving them will be apparent with reference to Examples, Test Examples, and Preparation Examples, which are described below in detail. However, the present invention is not limited to the examples, test examples, and preparations disclosed below, but may be embodied in various forms. The present invention is provided to fully inform those skilled in the art to which the present invention pertains, and the present invention is defined only by the scope of the claims.

Example  1: Schizandra, golden, Thawed blood  Each single extract preparation

<1-1> Single extract of Schizandra chinensis

50 g of Schisandra chinensis was thoroughly washed with water and dried in the shade. Dry Schizandra chinensis was added to the extraction container, and alcohol was extracted three times with 500 ml of alcohol. The extract was filtered through a filter paper, concentrated under reduced pressure using a vacuum rotary evaporator and lyophilized to prepare a powdery alcohol extract 12.3 g. The obtained alcohol extract was first fractionated with chloroform, and ethyl acetate acetate was added to the residue to fractionate to prepare 3.5 g of the final extract (hereinafter referred to as SCEA).

<1-2> sole extract of gold

50 g of gold was washed with water and dried in the shade. Dry golden was placed in the extraction container, and alcohol was extracted three times by adding 500 ml of alcohol. The extract was filtered through a filter paper, concentrated under reduced pressure using a vacuum rotary evaporator and freeze-dried to prepare 19.2 g of powdered alcohol extract. The obtained alcohol extract was first fractionated with chloroform, and ethyl acetate acetate was added to the residue to fractionate to prepare 1.7 g of final extract (hereinafter referred to as SBEA).

<1-3> Thawing blood  Sole extract manufacturer

50 g of thawed skin was washed with water and dried in the shade. The dried thawed blood was placed in an extraction container, and alcohol was extracted three times with 500 ml of alcohol. The extract was filtered through a filter paper, concentrated under reduced pressure using a vacuum rotary evaporator and lyophilized to prepare 5.9 g of a powdered alcohol extract. The obtained alcohol extract was first fractionated with chloroform and ethyl acetate solvent was added to the residue to fractionate to prepare 0.2 g of final extract (hereinafter referred to as KPEA).

Example  2: Preparation of Comparative Mixed Extract

<2-1> Preparation of Schisandra chinensis and Golden Mixed Extract

25 g each of Schizandra chinensis and gold was washed with water, and then dried in the shade. Dry Schizandra chinensis (Gold) was placed in an extraction container, and alcohol was extracted three times with 500 ml of alcohol. The extract was filtered through a filter paper, concentrated under reduced pressure using a vacuum rotary evaporator and lyophilized to prepare 22.1 g of a powdered alcohol extract. The obtained alcohol extract was first fractionated with chloroform and ethyl acetate solvent was added to the residue to fractionate to prepare 1.9 g of the final mixed extract (hereinafter referred to as SSEA).

<2-2> Schizandra and Thawed blood  Preparation of Mixed Extract

25g each of Schisandra chinensis and thawed skin was washed with water and dried in the shade. Dried Schisandra chinensis and thawed blood were placed in an extraction container, and alcohol was extracted three times with 500 ml of alcohol. The extract was filtered through a filter paper, concentrated under reduced pressure using a vacuum rotary evaporator, and lyophilized to prepare 14.8 g of an alcohol extract in powder form. The obtained alcohol extract was first fractionated with chloroform and ethyl acetate solvent was added to the residue to fractionate to prepare 1.7 g of the final mixed extract (hereinafter referred to as SPEA).

Example  3: (Schisandra, golden and Thawed blood ) mix Herbal medicine  Extract manufacturer

40 g of Schisandra chinensis, 20 g of gold, and 40 g of thawed blood were washed with water, and then dried in the shade. Dried Schisandra chinensis, golden and thawed blood were placed in an extraction container, and 1 liter of alcohol was added to extract alcohol three times. The extract was filtered through a filter paper and concentrated under reduced pressure using a vacuum rotary evaporator and lyophilized to prepare 31.5 g of an alcohol extract in powder form. The obtained alcohol extract was firstly fractionated with chloroform and ethyl acetate solvent was added to the residue to fractionate to prepare 2.9 g of the final mixed extract (hereinafter referred to as YD101).

Experimental Example  One: Hyaluronidase  ( hyaluronidase ) Activity inhibition

In order to determine the degree of inhibition of hyaluronidase activity of the extracts of Examples 1 and 3 of the present invention with respect to joint protection, the activity was measured using a hyaluronic acid digestion assay (Richman and Baer; Anal Biochem, 109, p376-381, 1980). Since hyaluronic acid is coupled to a large amount of water as mentioned above and is engaged in lubrication of the joints, the hyaluronic acid should not be decomposed so that the joints can be protected by active lubrication. The experiment is as follows.

2 mg / ml of hyaluronic acid dissolved in 0.05M sodium citrate buffer (pH 5.3) containing 0.15M sodium chloride and 0.02% (w / v) sodium azide and 3% (w / v) Agarose was mixed and hardened to the plate. A hole of 2 mm was made in the hardened agarose gel, and 5 μl of hyaluronidase (5,000 unit / mL) and 5 μl of sample (10 mg / mL) were added to the hole and incubated at 37 ° C. for 20 hours. After completion of the culture, 10% cetylpyridinium aqueous solution was poured into the gel, and then the hyaluronidase enzyme activity inhibitory activity was measured as the size of the clear zone.

As a result, as shown in Figure 1, the extract of Schizandra chinensis (SCEA) showed the inhibitory activity against hyaluronidase, but the extract of each of the gold (SCEA) and thawed skin (KPEA) did not show activity. On the other hand, YD101, a mixed herbal extract consisting of Schisandra chinensis, golden and thawed blood of the present invention, showed a clear synergic effect.

Experimental Example  2: Collagenase  ( collagenase ) Activity inhibition

A 1-well agarose solution and 0.15% gelatin (porcine gelatin) solution dissolved in collagenase buffer (50 mM Tris-HCl, 10 mM CaCl ₂ , 0.15 M NaCl, pH 7.8) were prepared. Ml / well) was hardened at room temperature for 1 hour. Extract sample 1 prepared for each concentration was incubated with 10 μl of collagenase-1 (0.1 mg / ml) for 1 hour. The reaction solution 10 was absorbed into a paper disk (6 mm), and then placed on the gel of each well and incubated at 37 ° C. for 18 hours. After completion of the reaction, the disc was removed from the gel and the gel was stained with Coomassie blue reagent. After removing the dyeing reagent, the inhibitory activity of collagenase enzyme activity was measured as the size of the formed (clear zone).

Experimental results, as shown in Figure 2, SCEA, SBEA, KPEA alone extracts of each of Schisandra chinensis, Golden, thaw skin did not have a large inhibitory activity of collagenase, whereas YD101 (Schisandra + Golden + mixed herbal medicine extract of the present invention) It was confirmed that thawed skin had a synergistic effect in a concentration-dependent manner at a concentration of 200 µg / ml or more.

Experimental Example  3: Elastase  ( elastase ) Activity inhibition

In order to evaluate the inhibitory effect of chondrogenic degradation of extracts in arthritis, the inhibitory activity against elastase, one of leukocyte granulase involved in joint destruction, was measured. Human leukocyte elastase enzyme was dissolved in 50 mM acetic acid buffer solution (pH 5.3) to prepare a 1 unit / ml solution. As an enzyme substrate, para-nitroanilide was dissolved in DMSO to prepare a 20 mM solution, which was used as a substrate solution for elastase enzyme. After mixing elastase and extracts, 10 µl was dispensed into a 96-well plate, dissolved in 10 mM sodium phosphate buffer solution (pH 6.8), and 200 µl of 200 paranitroamilide was added thereto. After 2 hours of incubation at the absorbance was measured at 410nm.

As a result, as shown in Figure 3 of the present invention, YD101 (Schisandra + Golden + thawed bark) of the mixed herbal medicine extract of the present invention showed a high activity inhibitory effect of more than 85%.

Experimental Example  4: articular cartilage protection experiment

Articular cartilage consists of water (70-80%), collagen (10-15%), proteoglycans (5-10%), and chondrocytes for lubrication and growth, and proteoglycans It has a structure in which several glycosaminoglycans (GAGs) are attached to a core protein. If the cartilage cells fail to produce proteoglycans or collagen properly due to external stimulation or the like, the cartilage deteriorates and the cartilage is damaged and arthritis is induced. In order to confirm the proteoglycan degradation inhibition and anti-inflammatory effects of the mixed herbal medicine extract of the present invention YD101 was carried out as follows.

<4-1> Cartilage Tissue and Cell Culture

Cartilage tissue isolated from rabbit joints was treated with 5% fetal bovine serum (fetal bovine serum, FBS, GIBCO BRL, USA) and penicillin-streptomycin 100 unit / ml inactivated by heat treatment (DMEM; Dulbeco's modified eagle's medium, GIBCO BRL, USA). Cartilage tissue was washed several times with the medium and incubated in a humidified 5% CO 2 incubator at 37 ° C. After 1-2 days the medium was replaced with basal medium containing 10% fetal calf serum deactivated by heat treatment and 10 mM HEPES, 100 units / ml penicillin-streptomycin, and 30 articular tissues were transferred to 48-well plates. After incubation for 1 hour, 10 ng / ml of interleukin-1beta (IL-1β R & D system, USA) was added to the medium to induce inflammation, and the extract 100-200 of the present invention was incubated at 37 ° C after treatment. After incubation for 3-7 days, the supernatant was collected, which was used as a sample of the following experiment while being stored at -20 ° C.

<4-2> Glycosaminoglycans  ( GAG Decomposition degree measurement

In order to determine the protective effect of the articular cartilage tissue of human and rabbit of the YD101 of the present invention, glycosaminoglycan (1,9-dimethylmethylene blue; The following experiment was conducted to determine the degree of degradation of GAG) (Frenc, h MM et al; Ann Biomed Eng, 32, pp 50-56, 2004). The concentration of GAG in the culture medium of articular cartilage tissue obtained by the method of Example <4-1> was measured by the amount of polyanionic material produced by reaction with a Blyscan dye solution, and standard Chondroitin sulfate was used as the material. 100 µl of each of the culture medium obtained in Experimental Example <4-1> was mixed with a dye solution, reacted at room temperature for 30 minutes, and centrifuged at 12,000 rpm for 10 minutes. After dissolving the precipitate in a Blyscan dye-dissociation solution, the spectroscopic GAG amount was measured at 540 nm, and the inhibition rate was calculated based on the amount of GAG degradation induced by interleukin-1beta (IL-1β).

Experimental results As shown in Figure 4, YD101, the mixed herbal medicine extract of the present invention inhibits GAG degradation into the medium at 100 ㎍ / ㎖ concentration treatment lower than 200 ㎍ / ㎖ SCEA (Schisandra chinensis) extract addition , IL-1beta (IL-1β) -induced cartilage cell degradation was confirmed to be significantly inhibited.

<4-3> PGE2  Ingredient analysis

The amount of PGE2 in the tissue culture supernatant of Experimental Example <4-1> was changed to PGE2. Measurement was performed using an enzyme immunoassay kit (PGE2 EIA kit, Cayman chemical, Ann Arbor, Michigan, USA) (Dovedi SJ et al., J. Urol., 174 (1), pp332-337, 2005). The culture supernatant was diluted 1:50 using a phosphate buffer solution, and 50 µl aliquots were dispensed, and a pre-coated plate coated with goat anti-mouse prostaglandin E2 monoclonal IgG. ), And the amount of synthesized PGE2 (synthesized PGE2) is shown in FIG. 5 by quantitative measurement after serial dilution of standard PGE2.

The mixed herbal extract of the present invention YD101 (Schisandra + Golden + Thawed Peel) significantly inhibited the production of PGE2 from cartilage tissue when treated at a lower concentration of 50-100 µg / ml than when 200 µg / ml SCEA (Schisandra chinensis) extract was added. As a result, the anti-inflammatory effect and analgesic mechanism of YD101 were confirmed.

Experimental Example  5: TPA  Induction mouse Edema  Experiment

Six-week-old ICR mice, which were fasted for 18 hours, were isolated for each experimental group, and after 1 hour of oral administration of each sample at 300 mg / kg, TPA (2.5㎍ / 20μl) was dissolved in acetone, and then Edema caused edema. After 4 hours, the thickness of ear edema was measured for each experimental group, and the difference in the thickness of the edema of the left ear where the inflammation was induced and the left ear where the inflammation was not induced was measured.

As shown in Figure 6, YD101, the mixed herbal extract of the present invention showed a high edema inhibitory ability of more than 50% against TPA-induced mouse ear edema, acetaminophen and a single extract (SCEA, SBEA, KPEA) The synergic effect was found to be significantly higher in comparison with.

Experimental Example 6: Collagen - induced Evaluation of Anti-arthritis Efficacy Using Arthritis ( CIA ) Mouse Model

In order to confirm whether the mixed herbal medicine extract (YD101) of the present invention can inhibit the pathological progression of arthritis, the inventors performed an antiarthritis efficacy evaluation experiment using a collagen-induced arthritis (CIA) mouse model.

<6-1> mixed Herbal medicine  Oral Administration of Extracts

The comparative mixture of Example 2, SSEA (Schisandra + Golden), SPEA (Schisandra + thawed skin), and YD101 (Schisandra + Golden + thawed skin) of the mixed herbal medicine extract of the present invention was prepared by dissolving in 0.5% CMC (carboxymethylcellulose). . The prepared fraction extracts were administered once daily at 200 mg / kg / day, connected to the 1 ml syringe for 3 weeks, and then directly administered 200 μl into the mouth of the mouse. As a positive control, celecoxib (celecoxib, Pfizer Co., Ltd.), commercially available as an anti-inflammatory drug, was orally administered in the same manner.

<6-2> CIA  Mouse arthritis

Collagen-induced arthritis (CIA) model was prepared according to Chondrex's method to induce arthritis in 8-week-old DBA / 1J mice. After emulsion by mixing with bovine type 2 collagen (Freagen) and CFA (Freund's complete adjuvant), this solution was injected intradermally into the tail of the mouse to induce primary immunity (D0). On day 21 after the first immunization, bovine type 2 collagen was mixed with ICFA (Freund's incomplete adjuvant) and emulsified in the same manner as above, followed by intradermal injection into the tail to induce a secondary immune response (D21). On the 22nd day, 8 animals were divided into 4 groups and SSEA (Schisandra + Golden), SPEA (Schisandra + Golden Thawed Skin), and YD101 (Schisandra + Golden + Thawed Skin) were orally administered for 3 weeks until 42 days. One to two weeks after the second immunity, joint symptoms began to appear gradually. Once a week, the weight and score of the foot that caused the swelling and swelling were evaluated.On day 52, blood collection was performed, autopsy was performed, swelling of the foot was fixed and demineralized, and H & E (Hematoxyline & Eosin) staining was followed. It was.

The results of comparing the anti-arthritis efficacy in the mouse CIA model of the comparative mixture of SSEA (Schisandra chinensis), SPEA (Schisandra chinensis + thawed skin) and YD101, the mixed herbal medicine extract of the present invention, are shown in FIG.

As a result, arthritis symptom began to appear after 26 days after the first immunization, and it was remarkably induced on the 39th day, and the symptoms increased rapidly until 53 days. There was no significant difference between the 22 and 42 days of oral administration of each sample, but since the 49th day, the SSEA (Schisandra + Golden) and SPEA (Schisandra + thawed blood) groups continuously induced arthritis symptoms, but the present invention In the group administered YD101 (Schisandra chinensis + Golden + Thawed Peel), the mixed herbal medicine extract showed statistically significant anti-arthritis effect.

The antiarthritis efficacy of YD101 was compared with the control drug celecoxib (100 mg / kg). Arthritis symptoms started on day 26 in arthritis-induced and treated groups with 0.5% CMC. Symptoms developed until day 53 after days of rapid change in symptoms from 35 days to 46 days. It was confirmed that the arthritis index was maintained after 53 days in the oral administration of YD101, and as a positive drug control group, it showed an efficacy equal to or higher than that of the celecoxib (100 mg / kg) administration group, which is a COX-2 inhibitor. FIG. 9 is a photograph of left and right Fujis showing arthritis-induced symptoms in each group, and in the control group receiving 0.5% CMC, redness and swelling were induced from the toes to the heel of both Fujis. Individuals receiving celecoxib and YD101 did not significantly induce arthritis symptoms.

Figure 10 shows the histological analysis of the foot tissue isolated from each group of individuals. As a result of the analysis of 8 groups, the left and right cells and the Fuji were determined based on the degree of inflammation, bone resorption, osteophyte formation and cartilage damage. As a result of qualitative analysis, the arthritis induction control group which received 0.5% CMC showed 6.13 ± 3.4, celecoxib group showed 3.50 ± 4.2 and YD101 group showed 3.13 ± 3.0, which showed excellent anti-arthritis effect comparable to celecoxib. .

From the results of the experimental example, YD101, the mixed herbal medicine extract of the present invention, showed excellent inhibitory activity against hyaluronidase, collagenase, and elastase enzymes, which are representative cartilage matrix-degrading enzymes that cause osteoarthritis.

In particular, the synergistic effect of the mixed herbal medicine extract (YD101) of the present invention in hyaluronidase was clearly confirmed. The mixed herbal extract of the present invention (YD101) significantly inhibited the degradation of cartilage matrix by induction of IL-1β treatment in cartilage tissue and chondrocyte cultures obtained from rabbits, and also inhibited the production of PGE2, a representative mediator of inflammation. This suggests an excellent formulation potential for degenerative osteoarthritis.

In addition, it showed better edema inhibition than acetaminophen, and the CIA mouse rheumatoid model experiment showed an effect equivalent to that of celecoxib used as a COX-2 inhibitor. Therefore, the value as a prophylactic or therapeutic composition for degenerative osteoarthritis as well as rheumatoid arthritis disease was confirmed.

Production Example  1: mixed Herbal medicine  Of extract Powder  Produce

20 mg of the mixed herbal medicine extract of Example 3,

 -100 mg of lactose,

Talc 10mg

Powder is prepared by mixing the above ingredients and filling into airtight cloth.

Production Example  2: mixed Herbal medicine  Injection Preparation of Extracts

-10 mg mixed herbal extract of Example 3

-Mannitol 180mg

-Sterile distilled water for injection 2974 mg

Na2HPO4,12H2O 26mg

Prepared in the content of Preparation Example 2 above per ampoules according to the conventional method for preparing injections

Production Example  3: mixed Herbal medicine  Of extract Health drink  Produce

-1000 mg mixed herbal extract of Example 3

-Citric acid 1000mg

-100g oligosaccharide

-Plum concentrate 2g

-1 g of taurine

-900ml total by adding purified water

After mixing the above components according to the conventional healthy beverage production method, and then 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 refrigerated and then stored in the present invention Used to prepare healthy beverage compositions.

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.

Claims (10)

A composition for the prevention or treatment of degenerative arthritis, comprising a mixed herbal extract of Schisandra chinensis, golden and thawed skin as an active ingredient.
The method of claim 1,
The extract is a composition characterized in that Schisandra chinensis, golden and thawed blood are mixed in a weight ratio of 2: 1: 2.
The method of claim 1,
Wherein said composition is a pharmaceutical composition.
delete A dietary supplement for preventing or improving degenerative arthritis, comprising a mixed herbal extract of Schisandra chinensis, golden and thawed skin.
The method of claim 5,
The food is a health functional food, characterized in that selected from the group consisting of beverages, meat, chocolate, food, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes and dietary supplements.
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