KR102543158B1 - Composition for preventing or treating inflammatory diseases or pain - Google Patents

Abstract

The present invention relates to a composition for preventing, treating, or improving inflammatory diseases or pain, and the composition containing, as an active ingredient, a composite extract of Samchil and Sukjihwang in which the weight ratio of ginsenoside Rg3 to ginsenoside Rb1 is 0.20 or more, the composition is anti-inflammatory, Not only is it more excellent in analgesic effect, but also has a very excellent effect in treating arthritis or periodontal disease, so it can be usefully used in pharmaceutical compositions or health functional foods for preventing, treating, or improving inflammatory diseases or pain.

Description

Composition for preventing or treating inflammatory diseases or pain {Composition for preventing or treating inflammatory diseases or pain}

The present invention relates to a composition for preventing or treating inflammatory diseases or pain, and more specifically, an inflammatory disease comprising as an active ingredient a composite extract of Samchil and Sukjihwang in which the weight ratio of ginsenoside Rg3 to ginsenoside Rb1 is 0.20 or more; It relates to a composition for preventing or treating pain.

Although it is well known that non-steroidal anti-inflammatory drugs (NSAIDs) such as phenylbutazone, diclofenac, and aceclofenac have excellent anti-inflammatory analgesic effects, many side effects occur when taken for a long time. That is also a well-known fact.

Therefore, as a substitute for these non-steroidal anti-inflammatory drugs, a lot of research has been conducted to prepare pharmaceutical preparations by extracting physiologically active ingredients effective for anti-inflammatory pain relief from oriental herbal medicines.

On the other hand, arthritis-related diseases are representative degenerative and intractable diseases from which about 12% of the world's population suffers, and there are about 2 million or more patients in Korea. Arthritis is a generic name for the appearance of systemic symptoms in the musculoskeletal system due to inflammatory changes in the musculoskeletal and connective tissues of the body.

The disease is characterized by chronic inflammation affecting the joints, bones, cartilage tissue or spinal cord, often causing permanent tissue damage, deformity, degeneration and disability (Hofbause, L. C., Heufelder, A. E.: The role of osteoprotegerin and receptor activator of nuclear factor kappaB ligand in the pathogenesis and treatment of rheumatoid arthritis, Arthritis and Rheumatism 44:253-259, 2001). Arthritis is classified as degenerative arthritis (osteoarthritis), rheumatoid arthritis, extra-articular rheumatism or collagen disease.

Osteoarthritis, which is the most common among arthritis diseases, is a disease in which local degenerative changes occur as articular cartilage wears away. Although the cause is uncertain, it is known to be a disease related to frailty or excessive weight, and in this disease, degenerative changes in articular cartilage appear primarily. Degenerative changes start in articular cartilage, cartilage cells become necrotic, and the matrix is destroyed by cathepsin B, cathepsin D, collagenase, and the like. The production of proteoglycan and collagen cannot keep up with the degree of destruction, and the ability of cartilage to adapt to external forces gradually decreases, eventually resulting in microfractures in the subchondral bone tissue. As the disease progresses, hardening of the subchondral bone, excessive formation of bone around the joint, deformation of the joint, etc. occur, and the surface of the cartilage becomes rough, and an inflammatory reaction appears repeatedly in the joint cavity surrounded by the articular membrane. Therefore, repetitive pain, stiffness of the joint, and gradual movement disorder of the joint are caused.

Among arthritis, rheumatoid arthritis is a systemic, chronic inflammatory disease in which pathological symptoms mainly appear symmetrically in movable joints, and is known as an autoimmune disease caused by an abnormality in the immune system, the cause of which has not yet been precisely identified. The disease is characterized by persistent inflammatory synovitis that causes cartilage destruction and bone erosion, resulting in structural deformity of the joint. Symptoms associated with rheumatoid arthritis include joint swelling, joint tenderness, inflammation, morning stiffness, and pain, especially when bending the joint. In a subject with advanced arthritis, bone erosion and structural damage including joint destruction occur (Firestein, G.S.: Evolving concept of rheumatoid arthritis, Nature 423:356-361, 2003). In addition, patients may have other clinical symptoms such as damage to various organs including damage to the skin, kidneys, heart, lungs, central nervous system and eyes due to vasculitis associated with autoimmune processes.

Currently used treatment methods for degenerative arthritis and rheumatoid arthritis are as follows. Drugs often used for initial treatment are non-steroidal anti-inflammatory drugs (NSAIDs). Although these NSAIDs slightly improve symptoms, they do not prevent joint cartilage loss or progression of the disease. When administered over a long period of time, severe side effects appear on the gastrointestinal system, central nervous system, hematopoietic organs, kidneys, and liver, and about half of patients using this treatment must discontinue treatment within one year (Langegger, T., Michel , B. A.: Drug treatment for rheumatoid arthritis., Clin Orthop. 366:22-30,1999). The next step is to take gold drugs (gold sodium thiomalate, gold sodium thiosulfate) or disease modifying anti-rheumatic drugs such as penicillamine and anti-malarials. DMARDs) are used. However, although these drugs also somewhat reduce the progression of arthritis, they can be accompanied by serious side effects, so only 5-15% of patients continue to use these drugs after 5 years of starting treatment with DMARD. If the above treatment is no longer effective, in some cases, the rheumatic joint must be replaced with an artificial joint by external arthroplasty (Simon L. S., Int. J. Clin. Pract., 54:243-249, 2000).

Therefore, there is an urgent need for the development of new substances effective for the treatment and prevention of arthritis with new actions and skeletal structure protection effects that minimize toxicity and side effects, which are limitations of current treatments for arthritis.

On the other hand, periodontal disease refers to a disease that occurs in the periodontium, which is composed of alveolar bone, gingiva, periodontal ligament, and cementum, and is limited to the gums. It is divided into gingivitis and periodontitis, in which the periodontal ligament and alveolar bone are destroyed due to severe gingivitis.

There are many causes of periodontal disease, but bacterial causes are the most representative. Immune system activated by lipopolysaccharide (LPS), a cell wall component of anaerobic gram-negative bacteria, or directly destroyed by cytotoxins such as hydrogen sulfide, ammonia, and toxic amines, which are metabolites of anaerobic gram-negative bacteria present around periodontal pockets Periodontal tissue destruction and inflammation are induced by various cytokines, such as active oxygen, prostaglandin, leukotriene, histamine, and interleukin secreted by the action of . In addition, collagen, which is the substrate of periodontal tissue, is decomposed by enzymes such as collagenase derived from bacteria and leukocytes, resulting in gum recession, and if left untreated, periodontal disease worsens. Therefore, antibacterial action against anaerobic gram-negative bacteria, which is a fundamental cause of periodontal disease, bacteriostatic action and removal of toxic products, and restoration of lost periodontal tissue are important factors in preventing and treating periodontal disease.

Currently, oral hygiene improvement of patients, surgical treatment (calculus removal, root planing, gingival curettage, periodontal tissue regeneration) and non-surgical treatment (pharmacotherapy) are used for the treatment of periodontal disease. Surgical treatment, which can be said to be the most effective method, is difficult to treat periodontal disease because of the inconvenience of having to visit the dentist for treatment and limitations such as visiting the dentist when the disease has progressed to a certain extent rather than preventing the disease. There is a problem that is not effective and most of them progress to chronic diseases. In addition, antibiotics used as an additional treatment have problems such as excessive drug delivery to unnecessary parts, which can cause side effects, and in addition, cases of isolated periodontal disease bacteria showing resistance have recently been reported.

In order to compensate for the limitations of such surgical treatment and the problems of using antibiotics and to improve the prevention and treatment effects of periodontal disease, it is necessary to develop pharmaceuticals having the effect of restoring destroyed and lost periodontal tissue.

Therefore, there is an urgent need to develop a composition for preventing or treating inflammatory diseases (eg, osteoarthritis, rheumatoid arthritis, periodontitis) or pain that can minimize the side effects of existing anti-inflammatory drugs, arthritis drugs, and periodontal disease drugs.

Hofbause, L. C., Heufelder, A. E.: The role of osteoprotegerin and receptor activator of nuclear factor kappa B ligand in the pathogenesis and treatment of rheumatoid arthritis, Arthritis and Rheumatism 44:253-259, 2001 Firestein, G.S.: Evolving concept of rheumatoid arthritis, Nature 423:356-361, 2003 Langenegger, T., Michel, B. A.: Drug treatment for rheumatoid arthritis., Clin Orthop. 366:22-30,1999 Simon L. S.: DMARDs in the treatment of rheumatoid arthritis: current agents and future developments., Int. J. Clin. Pract., 54:243-249, 2000

The present inventors found that a composition having a specific ratio of ginsenoside Rb1 and ginsenoside Rg3 by heat treatment of ginseng ginseng and ginsenoside Rg3 has anti-inflammatory activity as well as pain suppression effect in arthritis, cartilage protection effect, inflammatory cytokine expression inhibitory effect and periodontal The present invention was completed by confirming that the disease treatment effect was superior.

Accordingly, an object of the present invention is to provide a composition for preventing or treating inflammatory diseases or pain.

In addition, another object of the present invention is to provide a health functional food for preventing or improving inflammatory diseases or pain.

In order to achieve the above object, the present invention provides a composition for preventing or treating inflammatory diseases or pain, which contains, as an active ingredient, a composite extract of Samchil and Sukjihwang in which the weight ratio of ginsenoside Rg3 to ginsenoside Rb1 is 0.20 or more.

In the present invention, Samchil (三七, Notoginseng Radix Et Rhizoma) means the root and / or rhizome part of Samchil. Samchile is a perennial herb of the Druraceae family. It is smaller than ginseng and has 7 indigo leaves. The root is in the shape of a small silk drum. The plant is also called Samchil because it has 7 leaves attached to 3 branches, or Samchil ginseng because its shape is similar to Korean ginseng. The root contains 3 to 8% of saponin, the main components of which are ginsenosides Rb1, Rg1, Rd and notoginsenosides R1, R2, Fa, Fc, and a small amount of ginsenosides ( ginsenoside) F1, F2, Ra1, Ra2, Rb2, Rb3, Re, Rc, Rf, etc. are contained. It contains fewer essential oils than ginseng and also contains oleanolic acid. The root has hemostatic and cardiac action, and in animal experiments, it has been found that it has the effect of increasing the blood flow of the coronary artery, reducing the oxygen consumption of the myocardium, and reducing the lipid and cholesterol content in the blood.

In the present invention, Rehmanniae Radix Preparata is a steamed and dried root of a medicinal plant belonging to the Ginseng family, and is used as a medicine in oriental medicine. It is called Gujihwang, which is made by repeating steaming and drying 9 times, and its medicinal effect is the best.

In the present invention, the weight ratio of ginsenoside Rg3 to ginsenoside Rb1 may be 0.20 to 30, preferably 0.20 to 25. More preferably, it is 0.20 to 21.

In the present invention, the composite extract may further contain ginsenosides Rd, Rh1, Rh4, Rk1, Rk3 and Rg5 in addition to ginsenosides Rb1 and Rg3.

In the present invention, the weight ratio of the total content of ginsenosides Rh1, Rh4, Rk3, Rg5, Rk1 and Rd to ginsenoside Rb1 may be 1.1 to 75.

In the present invention, ginsenoside Rg3 may be included at 7.0% by weight or more of the total content of the ginsenosides Rb1, Rg3, Rd, Rh1, Rh4, Rk1, Rk3 and Rg5. In addition, the ginsenoside Rg3 may be included in an amount of 7.0 to 25.0% by weight, and may be included in an amount of 9.0 to 23.0% by weight.

In the present invention, the composite extract of Samchil and Sukjihwang may be extracted from a mixture of Samchil and Sukjihwang, or may be a mixture of a single extract of Samchil and a single extract of Sukjihwang. In addition, the composite extract is preferably an extract of a mixture of Samchil and Sukjihwang, but is not limited thereto.

In the present invention, the composite extract of the present invention can be extracted by conventional extraction methods, such as cold needles, warm needles, heating, etc., using crude drugs in their original form or in the form of shredded or powdered herbs. In the present invention, it is preferable to use Samchil in powder form, and in the case of Sukjihwang, it is preferable to use it as it is or by cutting it.

In addition, in the present invention, the extraction solvent may be water, alcohol or a mixed solvent thereof, preferably using a water / alcohol mixed solvent. The water/alcohol mixed solvent may be a 30 to 95 volume (v/v) % alcohol aqueous solution, preferably a 50 to 70 volume (v/v) % alcohol aqueous solution, and more preferably, a 50 volume (v/v) % alcohol solution. v/v) % alcohol aqueous solution. In addition, the alcohol may be methanol, ethanol or isopropanol, preferably methanol or ethanol, more preferably ethanol.

Specifically, samchil, sukjihwang or a mixture thereof is mixed in a water/alcohol mixed solvent, preferably a water/methanol mixed solvent or a water/ethanol mixed solvent, more preferably a water/ethanol mixed solvent at 70 to 85° C. for 2 to 4 An extract can be obtained by extracting for a period of time. In order to increase the extraction efficiency of the herbal medicine, the above process may be repeated several times or more, and the concentration of the extract may be concentrated under vacuum at 60 ° C or less, although it is not particularly limited. At this time, by treating the concentrate with heat treatment, acid treatment, enzyme treatment, ultrasonic treatment, etc., a composition having a weight ratio of ginsenoside Rg3 to ginsenoside Rb1 of 0.20 or more can be obtained.

According to one embodiment of the present invention, the composite extract of the present invention may be extracted from a mixture of Samchil and Sukjihwang. At this time, the weight ratio of Samchil and Sukjihwang included in the mixture may be 3: 1 to 18: 1. Preferably, the weight ratio may be 4:1 to 16:1, more preferably 8:1 to 16:1, and most preferably 8:1.

According to another embodiment of the present invention, the complex extract may be a mixture of a single extract of Samchil and a single extract of Sukjihwang. When preparing the extract of Samchil alone or the extract of Sukjihwang alone, it is preferable to extract using a water/alcohol mixed solvent, preferably a water/methanol mixed solvent or a water/ethanol mixed solvent, more preferably a water/ethanol mixed solvent, After concentration, it can be treated with heat treatment, acid treatment, enzyme treatment, sonication, etc. to prepare a single extract of Samchil or a single extract of Sukjihwang. At this time, if the weight ratio of ginsenoside Rg3 to ginsenoside Rb1 can be 0.20 or more in the mixture of ginseng extract alone and sukjihwang extract alone, after concentration, heat treatment, acid treatment, enzyme Processes such as treatment and sonication can be omitted.

In addition, when the single extract of Samchil and the single extract of Sujihwang are prepared using the same weight of Samchil and Sujihwang, respectively, the weight ratio of the mixture of the single extract of Samchil and the single extract of Sujihwang may be 1.5 to 9:1, preferably 3 to 9:1. It may be 6:1, and more preferably 3:1.

In addition, since the yield of Samchil alone extract and Sukjihwang alone extract may vary depending on the extraction method, it is preferable to adjust the mixing ratio of the original herbal medicine in consideration of the yield so that the weight ratio of Samchil and Sukjihwang is similar to that of the mixture of Samchil and Sukjihwang. It may be 3:1 to 18:1, preferably 4:1 to 16:1, more preferably 8:1 to 16:1, and most preferably 8:1.

The method for preparing the composite extract of Samchil and Sukjihwang of the present invention in more detail is as follows.

Samchil was powdered and used. If it is not powdered, the extraction efficiency of the active ingredient is reduced. Samchil powder and Sukjihwang each or a mixture thereof were prepared, and reflux extraction was performed at 75 to 85 ° C. for 4 to 6 hours using water or a water/alcohol mixture solution 5 to 10 times the weight of the crude drug as an extraction solvent, followed by filtration. do. Water or a water/alcohol mixture solution 5 to 10 times the weight of the herbal medicine is added to each residue, re-extracted by heating at 75 to 85 ° C, and filtered. Combine with the previous filtrate and concentrate to at least 10 brix, such as about 20 brix. If the amount of the extraction solvent is too small, it becomes difficult to stir and the solubility of the extract decreases, resulting in a decrease in extraction efficiency. If not, handling problems may occur.

The extract obtained by first and second extraction with water or a water/alcohol mixed solvent as described above is filtered and concentrated, and heat-treated at 90 ° C or higher to convert some of ginsenoside Rb1 to ginsenoside Rg3, thereby converting ginsenoside Rb1 to ginsenoside Rg3. A composite extract of Samchil and Sukjihwang in which the weight ratio of Rg3 to ginsenoside Rb1 is 0.20 or more can be obtained. The extract obtained in this way is additionally concentrated to obtain a soft extract form and freeze-dried to obtain a powdered extract, and an excipient is added and spray-dried to obtain a powdered extract.

In the present invention, the inflammatory disease may be arthritis or periodontal disease.

In the present invention, the arthritis may be degenerative arthritis (osteoarthritis) or rheumatoid arthritis.

In the present invention, the periodontal disease may be gingivitis or periodontitis.

In the present invention, room temperature means 15 to 25 °C.

In the present invention, prevention means any action that suppresses or delays an inflammatory disease or pain by administering the composition according to the present invention.

In the present invention, the composition is a known natural product or extract thereof known to be effective in inflammatory diseases or pain, or active functional substances, in addition to the complex extract of Samchil and Sukjihwang, in which the weight ratio of ginsenoside Rg3 to ginsenoside Rb1 is 0.20 or more. may further include. These components may be used independently or in combination.

In order to administer a pharmaceutical composition for preventing or treating inflammatory diseases or pain containing the complex extract according to the present invention as an active ingredient, it may be prepared by including at least one pharmaceutically acceptable carrier in addition to the active ingredient described above. there is. A pharmaceutically acceptable carrier may be a mixture of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components, and, if necessary, antioxidants and buffers , bacteriostatic agents and other conventional additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. Furthermore, using a method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA as an appropriate method in the art, it can be preferably formulated according to each disease or component.

The composition of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and the dosage may be the patient's weight, age, sex, health condition, The range varies according to diet, administration time, administration method, excretion rate, and severity of disease.

In the present invention, the composition of the present invention may be administered by oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, endothelial administration, intranasal administration, intrapulmonary administration, intrarectal administration, intracavitary administration, intrathecal administration, Not limited to this.

The daily dosage is 0.1 to 1000.0 mg/kg, preferably 0.1 to 500.0 mg/kg, based on the complex extract of Samchil and Sukjihwang of the present invention when administered orally, and it is more preferable to divide the administration once or several times a day. .

The present invention may be provided in the form of an oral composition for effective prevention and treatment of inflammatory diseases or pain prevention or treatment, and the dosage form of the oral composition is not particularly limited and may have a conventional dosage form. Specifically, it may have a formulation such as toothpaste, mouthwash or mouthwash. The oral composition provided in the present invention may contain various bases and additives necessary for formulation according to the formulation, and the type and amount of these components can be easily selected by those skilled in the art. For example, when the formulation of the oral composition is toothpaste, an abrasive, a wetting agent, a foaming agent, a binder, a sweetener, a pH adjusting agent, a preservative, an active ingredient, a flavoring agent, a brightening agent, a coloring agent, a solvent, and the like may be added.

Known natural products or extracts thereof known to have an effect on the inflammatory disease or pain are guaruegun extract, tokhal extract, hawthorn extract, hyssop extract, thawing hull extract, dermal extract, sagittarius extract, fennel root extract, raspberry extract, balsam extract, black It may be a gold head extract, a horse chestnut extract, or a mokcheon lyoja extract.

The ratio of the known natural substance or its extract known to have an effect on the inflammatory disease or pain and the active functional substance is not so important, but is generally selected in the range of 1 to about 90 parts by weight per 100 parts by weight of the composition of the present invention.

In order to achieve the above object, the present invention provides a health functional food for preventing or improving inflammatory diseases or pain, containing as an active ingredient a complex extract of Samchil and Sukjihwang in which the weight ratio of ginsenoside Rg3 to ginsenoside Rb1 is 0.20 or more. do.

Improvement in the present invention refers to any action that improves or beneficially alters inflammatory diseases or pains by administration of the composition according to the present invention.

The health functional food of the present invention includes food defined in the Korean Health Functional Food Act enacted on August 26, 2002 (FUNCTIONAL HEALTH FOODS ACT). Specifically, “health functional food” includes foods manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients having useful functionalities for the human body.

The health functional food for preventing or improving the inflammatory disease or pain may further include additives acceptable in food science.

The composite extract of Samchil and Sukjihwang, having a weight ratio of ginsenoside Rg3 to ginsenoside Rb1 according to the present invention of 0.20 or more, can be added to health functional foods for the purpose of improving inflammatory diseases or pain, and the extract according to the present invention When used as a food additive, the mixed herbal medicine extract may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, the extract according to the present invention is added in an amount of 100% by weight or less, preferably 50% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range. .

There is no particular limitation on the type of food. Examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and includes all health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The aforementioned natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrate is generally about 0.1 to 20.0 g, preferably about 1 to 10.0 g per 100 ml of the composition of the present invention.

In addition to the above, the health functional food of the present invention is various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol , carbonation agents used in carbonated beverages, and the like. In addition, the composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not critical, but is generally selected in the range of 0.05 to 50 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the present invention relates to a composition for preventing or treating inflammatory diseases or pains containing as an active ingredient a composite extract of Samchil and Sukjihwang having a weight ratio of ginsenoside Rg3 to ginsenoside Rb1 of 0.20 or more, including a mammal in need thereof Provided is a method for preventing or treating an inflammatory disease or pain by administering to a subject.

The present invention provides a use of a composite extract of Samchil and Sukjihwang in which the weight ratio of ginsenoside Rg3 to ginsenoside Rb1 is 0.20 or more for preparing a pharmaceutical preparation for preventing or treating inflammatory diseases or pain.

The composition according to the present invention has excellent anti-inflammatory and analgesic effects as well as excellent treatment effects for arthritis or periodontal disease, so it can be usefully used in pharmaceutical compositions or health functional foods for preventing, treating, or improving inflammatory diseases or pain. .

1a to 1b show high-performance liquid chromatogram results showing the component profiles of the composite extracts of Samchil and Sukjihwang of Preparation Example 1-1 and Comparative Preparation Example 1, respectively.
Figure 2 shows the effect of the combined extract of Samchil and Sukjihwang of the present invention on the inhibition of cartilage destruction in the MIA-induced arthritis model as a photograph of the knee joint pathological tissue. In FIG. 2, A is the normal control group (NC group), B is the MIA group, C is the Celecoxib group, D is Preparation Example 1-1 group, and E is Comparative Preparation Example 1 group, respectively.
Figure 3 is a diagram showing the effect of the composite extract of Samchil and Sukjihwang on the arthritis index in the type 2 collagen-induced arthritis model of the present invention. In FIG. 3, 1 represents the normal group, 2 represents the control group, 3 represents the 150 mg/kg group of Preparation Example 1-1, 4 represents the 300 mg/kg group of Preparation Example 1-1, and 5 represents 100 mg/kg of celecoxib.
Figure 4 is a diagram showing the effect of the composite extract of Samchil and Sukjihwang of the present invention on improving clinical attachment level (CAL) in a ligation-induced periodontal disease model. In Figure 4, 1 is a normal control group, 2 is a negative control group, 3 is Preparation Example 1-1 7.5 mg/kg administration group, 4 is Preparation Example 1-1 30 mg/kg administration group, 5 is Preparation Example 1-1 90 mg/kg Each kg administration group is shown.
Figure 5 is a diagram showing the effect of the composite extract of Samchil and Sukjihwang of the present invention on the inhibition of fever pain. In Figure 5, 1 is the control group, 2 is Preparation Example 1-1 25 mg / kg group, 3 is Preparation Example 1-1 100 mg / kg group, 4 is Preparation Example 1-1 300 mg / kg group, 5 is celecoxib Each of the 50 mg/kg groups is shown.
Figure 6 is a diagram showing the tail-flick test results of the composite extract of Samchil and Sukjihwang of the present invention. In Figure 6, 1 is the control group, 2 is Preparation Example 1-1 50 mg / kg group, 3 is Preparation Example 1-1 200 mg / kg group, 4 is Preparation Example 1-1 600 mg / kg group, 5 is celecoxib Each of the 100 mg/kg groups is shown.
Figure 7 is a diagram showing the results of the acetic acid-writhing test of the composite extract of Samchil and Sukjihwang of the present invention. In Figure 7, 1 is the control group, 2 is Preparation Example 1-1 50 mg / kg group, 3 is Preparation Example 1-1 200 mg / kg group, 4 is Preparation Example 1-1 600 mg / kg group, 5 is celecoxib Each of the 100 mg/kg groups is shown.
Figure 8 is a diagram showing the rotarod test results of the composite extract of Samchil and Sukjihwang of the present invention. 8, 1 is a rat control group, 2 is Preparation Example 1-1 100 mg/kg group, 3 is Preparation Example 1-1 300 mg/kg group, 4 is a mouse control group, 5 is Preparation Example 1-1 300 mg/kg group, 6 indicates Production Example 1-1 600 mg/kg group, respectively.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are not intended to limit the scope of the present invention, which should be interpreted to aid understanding of the present invention.

manufacturing example 1. Preparation of a composite extract of Samchil and Sukjihwang (extract of a mixture of Samchil and Sukjihwang)

manufacturing example 1-1. Preparation of complex extracts of Samchil and Sukjihwang

Samchil was purchased from Myohyang Samchil Co., Ltd. and Pharmaline Co., Ltd. from China. Jihwang Sook purchased and used products distributed in Korea through Pure Mind. After checking the presence or absence of foreign substances for each herbal medicine, Samchil and Sukjihwang, only clean herbal medicines were selected and used in the experiment. Samchil was finely ground and used. That is, Samchil powder was used. Sukjihwang was used in its original form. Samchil 8: Put 540 g of the mixture in the weight ratio of Sukjihwang 1 into a 5 L flask, extract with 3.78 L of 50% (v/v) ethanol at 84 ° C. while stirring at reflux for 4 hours, filter, and leave the residue Extraction was performed with 2.7 L of 50% (v/v) ethanol at 84° C. for 2 hours under reflux stirring. The extract was concentrated to a concentration of about 20 brix and then refluxed at 100 °C for 16 hours. After filtering this again, it was concentrated by vacuum concentrating using a rotary vacuum evaporator (N-12, EYELA) at 60 ° C or less, and then lyophilized to powder and used in the experiment. The yield was 30%.

manufacturing example 1-2. Preparation of complex extracts of Samchil and Sukjihwang

Preparation Example 1-2 was prepared in the same manner as in Preparation Example 1-1, except that reflux was performed at 100 ° C. for 8 hours instead of 16 hours in Preparation Example 1-1.

Preparation Example 1-3. Preparation of complex extracts of Samchil and Sukjihwang

Preparation Example 1-3 was prepared in the same manner as in Preparation Example 1-1, except that reflux was performed at 100 ° C. for 32 hours instead of 16 hours in Preparation Example 1-1.

Preparation Example 1-4. Preparation of complex extracts of Samchil and Sukjihwang

Preparation Example 1-4 was prepared in the same manner as in Preparation Example 1-1, except that reflux was performed at 100 ° C. for 64 hours instead of 16 hours in Preparation Example 1-1.

Preparation Example 1-5. Preparation of complex extracts of Samchil and Sukjihwang

Preparation Example 1-5 was prepared in the same manner as in Preparation Example 1-1, except that in Preparation Example 1-1, the weight ratio of Samchil powder and Sukjihwang was 16: 1 instead of 8: 1.

Preparation Example 1-6. Preparation of complex extracts of Samchil and Sukjihwang

Preparation Example 1-6 was prepared in the same manner as in Preparation Example 1-1, except that in Preparation Example 1-1, the weight ratio of Samchil powder and Sukjihwang was 4: 1 instead of 8: 1.

Preparation Example 2. Preparation of a composite extract of Samchil and Sukjihwang (a mixture of a single extract of Samchil and a single extract of Sukjihwang)

Preparation Example 2-1. Preparation of complex extracts of Samchil and Sukjihwang

Preparation of extract of Samchil alone

Samchil was finely ground and used. 200 g of Samchil was put in a 3 L flask, extracted with 1.4 L of 50% (v/v) ethanol at 84 ° C. while stirring at reflux for 4 hours, filtered, and 1.0 L of 50% (v/v) ethanol was added to the residue. The mixture was extracted with reflux stirring at 84 °C for 2 hours using ethanol. The extract was collected, filtered, concentrated under reduced pressure to about 20 brix, and then refluxed at 100° C. for 16 hours. After filtering again, it was concentrated under vacuum using a rotary vacuum evaporator (N-12, EYELA) at 60 ° C or less, freeze-dried, and powdered to be used in experiments. The yield was 25.0%.

Preparation of Sukjihwang extract alone

Sukjihwang was used in its original form. Put 200 g of Suk Rehmannia Root in a 3 L flask, extract with 1.4 L of 50% (v/v) ethanol at 84 ° C. while stirring at reflux for 4 hours, filter, and add 1.0 L of 50% (v/v) to the residue. The mixture was extracted with reflux stirring at 84 °C for 2 hours using ethanol. After collecting and filtering the extract, concentrated in vacuum using a rotary vacuum evaporator (N-12, EYELA) at 60 ° C or lower, freeze-dried, powdered, and used in experiments. The yield was 67%.

Preparation of a mixture of Samchil alone extract and Sukjihwang alone extract

A composite extract of Samchil and Sukjihwang was prepared by mixing the single extract of Samchil and the single extract of Sukjihwang prepared as described above at a weight ratio of 6: 1.

Preparation Example 2-2. Preparation of complex extracts of Samchil and Sukjihwang

A composite extract of Samchil and Sukjihwang was prepared by mixing the single extract of Samchil and the single extract of Sukjihwang prepared as described in Preparation Example 2-1 at a weight ratio of 3:1.

Comparative Preparation Example 1. Preparation of a composite extract of Samchil and Sukjihwang

After checking the presence or absence of foreign substances for each herbal medicine, Samchil and Sukjihwang, only clean herbal medicines were selected and used in the experiment. Samchil was finely ground and used. Sukjihwang was used in its original form. Samchil 8: Put 540 g of the mixture in the weight ratio of Sukjihwang 1 into a 5 L flask, extract with 3.78 L of 50% (v/v) ethanol at 84 ° C. while stirring at reflux for 4 hours, filter, and leave the residue Extraction was performed with 2.7 L of 50% (v/v) ethanol at 84° C. for 2 hours under reflux. After collecting and filtering the extract, concentrated in vacuum using a rotary vacuum evaporator (N-12, EYELA) at 60 ° C or lower, freeze-dried, powdered, and used in experiments. The yield was 29.7%.

Comparative Preparation Example 2. Preparation of composite extracts of Samchil and Sukjihwang

Comparative Preparation Example 5 was prepared in the same manner as in Preparation Example 1-1, except that reflux was performed at 100 ° C. for 2 hours instead of 16 hours in Preparation Example 1-1.

Comparative Preparation Example 3. Preparation of a composite extract of Samchil and Sukjihwang

Comparative Preparation Example 6 was prepared in the same manner as in Preparation Example 1-1, except that reflux was performed at 100 ° C. for 4 hours instead of 16 hours in Preparation Example 1-1.

Test Example 1. Ginsenoside content analysis and chromatogram

Each of the composite extracts of Samchil and Sukjihwang of Preparation Examples 1 to 2 and Comparative Preparation Examples 1 to 3 were used as samples. Precisely weighed 500 mg of the sample, put 25 mL of water in a 50 mL volumetric flask, shake it occasionally for 1 hour, and then add 20 mL of methanol and shake it ultrasonically for 30 minutes. After bringing this solution to room temperature, it was leveled with methanol and filtered through a 0.45 μm membrane filter paper to be used as a test solution. Separately, about 10 mg of the ginsenoside Rb1 standard was precisely weighed and dissolved in methanol to make exactly 50 mL. 5 mL of this solution was put into a 10 mL volumetric flask, leveled with water, and filtered through a 0.45 μm membrane filter paper, and the solution was used as a standard solution. 20 μL of the sample and standard solutions were tested according to the liquid chromatography method under the following conditions, and the peak areas of the sample and standard solutions were measured. Standards of each indicator component were purchased from sigma-aldrich, chromadex, and embo research institutes. A mixed solvent of water and acetonitrile was used as a mobile phase using a Zorbax SB C18 (3.5 μm, 4.6 × 150 mm) column, and the ratio of organic solvent was gradually increased so that the acetonitrile ratio started at 1% and reached 80% in 40 minutes. . The measurement wavelength was 203 nm.

The results of high performance liquid chromatography (HPLC) analyzed by the above method are shown in FIGS. 1a to 1b and Table 1. Table 1 shows the content of ginsenosides in the composite extracts of Samchil and Suk Rehmannia Root of Preparation Examples 1 to 2 and Comparative Preparation Examples 1 to 3 measured by the high-performance liquid chromatography method, and various ginsenosides for ginsenoside Rb1 The relative content of the side was shown in Table 2 below.

Table 1. Ginsenoside content (mg/g)

Table 2. Ginsenosides Rb1 Relative content of various ginsenosides

As shown in Figure 1b, it was found that the composite extract of Samchil and Sukjihwang of Comparative Preparation Example 1, which was not subjected to separate heat treatment, did not contain ginsenoside Rg3. In addition, in the extracts of Samchil and Sukjihwang of Comparative Preparation Example 1, notoginsenoside R1 (notoginsenoside R1; NG.R1), ginsenoside Rg1 (G.Rg1), ginsenoside Rb1 (G.Rb1), ginsenoside It was found that Rd (G.Rd) was the main component.

However, when heat treatment is performed, as shown in FIG. 1a, ginsenoside Rg3 (G.Rg3) is generated, in addition to ginsenoside Rh1 (G.Rh1), ginsenoside Rh4 (G.Rh4), ginsenoside It was found that Rk3 (G.Rk3), ginsenoside Rg5 (G.Rg5), ginsenoside Rk1 (G.Rk1), and ginsenoside Rg2 (G.Rg2) were also produced.

test example 2. Acute toxicity test of complex extracts of Samchil and Sukjihwang

The herbal medicines used in the present invention are classified as food raw materials, so it is considered that there is no problem with the safety of the mixed herbal medicine extract. Acute toxicity experiments were conducted using 6-week-old SPF rats as experimental animals. In 10 animals per group, the composite extract of Samchil and Sukjihwang of Preparation Example 1-1 of the present invention was dissolved in 0.5% CMC (carboxymethyl cellulose), and the animals fasted for 17 hours were orally administered once at a dose of 5 g / kg. After the administration of the test substance, the death of the animal, clinical symptoms, and weight change were observed, and after autopsy, abnormalities in the abdominal and thoracic organs were observed with the naked eye.

As a result of the test, there were no unusual clinical symptoms or dead animals in all animals administered with the test substance, and no toxic changes were observed in weight change, blood test, and autopsy findings. As a result of the above, the mixed herbal medicine extract of the present invention did not show toxicity changes up to 5 g/kg in rats, and the minimum lethal dose (LD ₅₀ ) of oral administration was judged to be a safe substance of 5 g/kg or more.

test example 3. Chronic toxicity test of complex extracts of Samchil and Sukjihwang

The herbal medicines used in the present invention are classified as food raw materials, so it is considered that there is no problem with the safety of the mixed herbal medicine extract. A chronic toxicity test was conducted using 6-week-old specific pathogen-free (SPF) rats as experimental animals. In 10 animals per group, the complex extract of Samchil and Sukjihwang of Preparation Example 1-1 of the present invention was dissolved in 0.5% CMC, and the highest concentration was 2400 mg / kg and the azeotrope was 0.5. It was administered orally weekly. After administration of the test substance, mortality, clinical symptoms, drinking water intake, eye examination, and weight change of the animals were observed, and after autopsy, abnormalities in the abdominal and thoracic organs were observed with the naked eye. In addition, safety was evaluated by integrating data such as organ weight, hematological examination, blood biochemical examination, histopathology, and urine analysis.

In the repeated administration toxicity test, the test substance, Preparation Example 1-1, was administered orally to 6-week-old male and female Sprague-Dawley rats for 26 weeks to evaluate the toxic reaction and its safety, followed by a 4-week recovery period. was conducted to confirm the reversibility of toxic changes. A medium dose (1,200 mg/kg) and a low dose (600 mg/kg) were set with a test substance of 2,400 mg/kg as a high dose and an azeotrope of 0.5. 12 animals were set per group, and 6 animals were added to the high dose and control groups to form recovery groups. In this experiment, no deaths were observed, and during the test period, general symptoms, body weight, feed intake, ophthalmology test, urine test, and visual findings at autopsy, organ weight, hematological test, blood biochemical test, and pathology test in Manufacturing Example 1 No observed adverse effect level (NOAEL) was judged to be 2,400 mg/kg or more as no toxic reactions were observed associated with repeated administration of the complex extracts of Samchil and Sukjihwang of -1. If this is converted into a human dose in consideration of the body surface area, it corresponds to 23,339 mg/day·60kg·person.

Example 1. Confirmation of the inhibitory effect of NO, an inflammation-related indicator

In order to measure the effect of the composite extract of Samchil and Sukjihwang on NO production, which is an important indicator (factor) in inflammation, 2 × 10 ⁵ cells of RAW 264.7 cells, a mouse macrophage cell line, were dispensed in a 24-well plate, and the next day Preparation Example 1 to The complex extracts of Samchil and Sukjihwang of Preparation Example 2 and Comparative Preparation Examples 1 to 3 were treated with 100 μg/mL, and after 1 hour, lipopolysaccharide (LPS) was added so that the concentration was 2 μg/mL so that NO was produced. cells were activated. NO (Nitrite) standard included in the Griess reagent system kit (Promega, USA) was prepared at 100 μM using DMEM. After culturing for 24 hours, 50 μL of the medium collected from each well of the 24-well plate and the calibration standard solution were added to each well of the 96-well plate. 100 μM of NO (Nitrite) standard solution was diluted in half with DMEM to prepare standard solutions for calibration curves to be 50, 25, 12.5, 6.25, 3.13, and 1.56 μM. DMEM was used for 0 µM of the standard solution for the calibration curve. After putting 50 μL of the sulfanilamide solution included in the Griess reagent system kit into each well of a 96-well plate, it was blocked from light and allowed to stand at room temperature for 10 minutes. After putting 50 μL of the NED solution included in the Griess reagent system kit into each well of a 96-well plate, the light was blocked and left at room temperature for 10 minutes. Absorbance was measured at 540 nm by Multiskan spectrum. As a control group, an LPS-only treatment group was used. By measuring the amount of NO production, the production inhibition rate compared to the control group was calculated and shown in Table 3.

The composite extracts of Samchil and Sukjihwang of Preparation Examples 1 to 2 and Comparative Preparation Examples 1 to 3 were treated for each concentration and tested in the same manner as above, and the IC ₅₀ results are shown in Table 4.

Table 3. NO production inhibition rate

As shown in Tables 2 and 3, as a result of evaluating the NO production inhibitory activity for the composite extracts of Samchil and Sukjihwang with various weight ratios of ginsenoside Rg3 to ginsenoside Rb1, ginsenoside Rg3 has a The NO production inhibitory activity was remarkably high in the composite extracts of Samchil and Sukjihwang with a weight ratio of 0.20 or more to the total.

Table 4. NO production inhibition rate (IC ₅₀ )

As shown in Tables 2 and 4, the 50% inhibitory concentration (IC ₅₀ ) of NO when the composite extract of Samchil and Sujihwang of Preparation Example 1-1 in which the weight ratio of ginsenoside Rg3 to ginsenoside Rb1 is 0.20 or more is treated. As 126.4 μg/mL, compared to 180.8 μg/mL, which is a 50% inhibitory concentration of NO when treated with the composite extract of Samchil and Sukjihwang in Comparative Preparation Example 1, in which the weight ratio of ginsenoside Rg3 to ginsenoside Rb1 does not belong to 0.20 or more. remarkably low.

Example 2. Confirmation of anti-inflammatory, analgesic and chondroprotective effects in MIA-induced osteoarthritis model

2-1. Disease induction and administration of complex extracts of Samchil and Sukjihwang

MIA (Monosodium iodoacetate, I2512, Sigma, Poole, UK) was dissolved in saline for injection at a concentration of 60 mg/mL in 12-week-old Sprague dawley rats (Dae Han Bio Link CO., Chungbuk, Korea), and the animals were placed in an anesthesia chamber ( Matrix, US/VME-2) and anesthetized with diethyl ether, the MIA was inserted into the right knee joint using a 0.5-inch gauge needle (model 750; Hamilton Company, Reno, NV). 50 μL {MIA 3 mg/body (subject)} was injected via the infrapatellar ligament. A normal control group (Naive) was injected with 50 μL of saline at the same location of the right knee joint. Test substances (Preparation Example 1-1, Comparative Preparation Example 1, respectively) were dissolved in a 0.5% CMC solution, and then homogenized as much as possible using a sonicator before use. The test substance and control substance (Celecoxib, 20 mg/kg) were orally administered once a day for a total of 21 days from the 7th day (day 7) to the 28th day (day 27) after MIA injection. The normal control group (NC) and the osteoarthritis-induced group (MIA) were orally administered with a 0.5% CMC solvent once daily at a dose of 10 mL/kg for the same administration period as the test substance. The number of animals was 10 per group, and each test substance and control substance were administered at a fixed time every day. The test drug was administered at 25 mg/kg.

2-2. Plantar febrile hyperalgesia measurement (Plantar test)

The thermal nociception threshold was set by adapting the plantar part of the right hind paw at 30 ° C for 10 minutes using a thermal nociception device (IITC life science, USA), then raising it to 50 ° C, placing the rat on the scaffold and licking the hind paw or lifting it from the floor. The time at which the raising reaction appears (paw withdrawal latency, PWL) was measured. The reaction measurement time was limited to within 30 seconds to prevent tissue damage, and the average value was used by measuring three times. In the normal control group, MIA-induced group, and experimental group, measurements were taken at regular times before (day 7) and after administration of degenerative arthritis medication once a week (day 14, day 21, day 27) (Hargreaves K, Dubner R, Brown F, Flores). C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32: 77-88, 1988). The amount of change in PWL at each time point after drug administration compared to PWL before drug administration was calculated, and statistical processing was performed by student t-test (significance level p < 0.05, 0.01).

As can be seen in Table 5 below, the composite extract of Samchil and Sukjihwang of Preparation Example 1-1 had the best analgesic effect and was statistically significant compared to the MIA group. In addition, a statistically significant analgesic effect was shown when compared with the Celecoxib administration group. The composite extract of Samchil and Sukjihwang of Preparation Example 1-1 showed superior effects than the composite extract of Samchil and Sukjihwang of Comparative Preparation Example 1.

Table 5. Paw withdrawal latency, PWL ) change (second)

2-3. Analysis of inflammatory and anti-inflammatory cytokines in the blood

After fasting for 16 hours before autopsy, blood was collected from the abdominal aorta by laparotomy under diethyl ether anesthesia on the day of the experiment (day 28). The collected blood was left at room temperature for 30 minutes, put into a heparin-untreated centrifuge tube (SST tube), and centrifuged at 3000 rpm at 4 ℃ for 20 minutes to separate serum and stored at -70 ℃ until used in experiments. . The contents of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-10, which are known as inflammation-related cytokines, in serum were evaluated using the Enzyme-Linked ImmunoSorbent Assay (ELISA) kit (R&D Systems Inc. Minneapolis, USA) using a sandwich assay. ) was measured. For the quantification procedure of each cytokine, the protocol provided by the manufacturer was used. Student t-test was performed and statistically treated (significance level p < 0.05, 0.01).

As shown in Table 6 below, the concentrations of TNF-α and IL-1β in blood when the composite extracts of Samchil and Sukjihwang of Preparation Example 1-1 having a weight ratio of ginsenoside Rg3 to ginsenoside Rb1 were 0.20 or more were treated. The weight ratio of ginsenoside Rg3 to ginsenoside Rb1 was significantly lower than the blood concentrations of TNF-α and IL-1β in the case of treating the composite extract of Samchil and Sukjihwang of Comparative Preparation Example 1, which did not belong to 0.20 or more. In addition, the blood concentration of IL-10 in the case of treatment with the composite extract of Samchil and Sukjihwang in Preparation Example 1-1 having a weight ratio of ginsenoside Rg3 to ginsenoside Rb1 of 0.20 or more is ginsenoside Rg3 relative to ginsenoside Rb1. The blood concentration of IL-10 was significantly higher than that of the case where the composite extract of Samchil and Sukjihwang of Comparative Preparation Example 1, which did not have a weight ratio of 0.20 or more, was treated.

Table 6. Blood cytokine concentration ( pg /mL)

2-4. knee joint histopathology test

The right knee joint of each animal was necropsied on the end of the experiment and the knee joint was collected for histopathological examination. The right hip and tarsal joints were separated and extracted to prevent damage to the knee joints, and then the soft tissues around the proximal tibialis and distal femoral joints were trimmed with a sterilized surgical knife. The collected knee joints were fixed in 10% formalin for 24 hours, and decalcification was performed while exchanging the solution with 10% formic acid at 24 hour intervals for 72 hours before tissue processing. A sagittal section of the knee joint was made through a coronal section so that the articular surfaces of the medial condyles of the femur and tibia could be well identified. Using a tissue processor (VIP-5Jr, Sakura Finetek, Japan), general dehydration, clearing, and tissue processing processes are performed, paramin embedding is performed, and a rotary microtome (Rotary Microtome, RM2245, Leica 2040, DE) is used 5 μm-thick knee joint thin sections were prepared. Hematoxylin & Eosin staining and Safranin O staining were performed on each section for each individual. H&E staining: Knee joint thin sections were deparaffinized, hydrated, stained with hematoxylin and eosin, dehydrated and clarified, and then sealed. Tissue slides were observed using an optical microscope (Olympus BX51, Olympus Optical Co. Japan) for inflammatory cell infiltration, cartilage and bone destruction levels, and histopathological changes in articular cartilage. Safranin-O fast green (SOFG) staining: Knee joint sections were deparaffinized and hydrated, then reacted with Weigert's iron hematoxylin (HT110232, Sigma, St Louis, USA) solution for 10 minutes, washed with water, and 0.02% fastgreen ( FCF) solution (F7258, Sigma, St Louis, USA) for 5 minutes. After reacting with 1% acetic acid solution, it was stained with 0.1% Safranin O (S2255, Sigma, St Louis, USA) solution. The slides stained with Safranin O-fastgreen were washed for 5 minutes to make the nuclei a clear color, dehydrated and clarified, and then sealed. Damage to the cartilage tissue was confirmed using an optical microscope (Olympus BX51, Olympus Optical Co. Japan).

To evaluate the degree of progression of degenerative arthritis, the osteoarthritis index was quantified by classifying the degree of damage according to the histopathological observation of the depth and degree of articular cartilage damage according to the method of Bar-Yehuda et al. Student t-test was performed and statistically treated (significance level p < 0.05, 0.01).

On the histopathological findings of this example, all subjects in the NC group injected with intra-articular saline had a homogeneous cartilage layer and well-developed chondrocyte maturation and arrangement, and the synovial membrane of the joint was also covered with a single layer of synovial cells. Maintained a normal state was doing Upon Safranine-O staining, the entire layer of articular cartilage was stained dark purple red and was observed to be rich in proteoglycan content (see Fig. 2). On the other hand, in the MIA group, which caused osteoarthritis, the thickness of the cartilage layer was very thin, and most of the femoral articular cartilage, tibia articular cartilage, and patellar articular cartilage were degenerated and necrotic. Erosion progressed until, and granulation tissue accompanied by infiltration of inflammatory cells was present on the surface of the damaged cartilage tissue (see FIG. 2). In some individuals, along with the degeneration and degenerative findings of the thickened joint synovium and articular cartilage, the subchondral bone tissue was exposed into the joint cavity due to the exfoliation of the cartilage layer, and the reconstructive process was frequently observed due to fibrotic proliferation. Most of the articular cartilage layer of the MIA group was destroyed, and the Safranine-O stain was very lightly stained or not stained at all. In Comparative Preparation Example 1, most of the subjects had moderate damage in the middle zone of the articular cartilage and thinning, and the destruction of subchondral bone tissue and the degree of fibrous tissue proliferation were observed in a pattern similar to that of the lesion in the MIA group. . In addition, the degeneration of the articular cartilage layer was also observed depending on the site, and the effect of inhibiting the degenerative damage of the joint was weakly confirmed (see FIG. 2). Even on Safranine-O staining, only the marginal and deep parts of the articular cartilage were stained in a pale tone. On the other hand, in the Celecoxib group and Preparation Example 1-1 group, compared to the osteoarthritis-induced MIA group, the range of articular cartilage with degenerative necrosis was observed to be reduced, and the degree of exposure of subchondral bone tissue due to the exfoliation of the cartilage layer was also reduced. Fibrosis around bone tissue and synovial thickening due to proliferation of synovial cells were also weakly observed. Even in the case of Safranine-O staining, it was confirmed that the progression of osteoarthritis by MIA administration was effectively inhibited, as the stained range and staining image were increased compared to the MIA group (see FIG. 2).

After observing the right knee joint stained with H & E and Safranin-O fast green, based on this, the results of the osteoarthritis index according to the degree and depth of articular cartilage damage and histopathological findings in each group are shown in Table 7. The osteoarthritis index was 0.0 ± 0.00 points and 4.5 ± 0.85 points in the NC group and the MIA group, respectively. The test group was 3.1 ± 1.52 points in the Celecoxib group, 2.5 ± 0.85 points in Preparation Example 1-1 group, and 3.7 points in Comparative Example 1 group. It was confirmed as ± 1.34 points. Both the MIA group and the test group showed a significant increase in the osteoarthritis index (p < 0.01) compared to the normal control NC group, and among the test groups, the Celecoxib group (p < 0.05) and Preparation Example 1-1 group (p < 0.01) showed a significant decrease compared to the MIA group, indicating that the degree of damage to articular cartilage tissue was improved in the treatment dose of these test substances. Preparation Example 1-1 group showed the lowest arthritis index compared to the MIA group, so it was judged to most effectively suppress the degenerative change and damage progress of articular cartilage caused by MIA.

Table 7. Histological Arthritis Index

Example 3. Induce collagen rheumatism Efficacy evaluation in arthritis model

3-1. Induction of type 2 collagen rheumatism Arthritis lab animal

Male DBA1/1J mice aged 5 to 6 weeks were purchased from Central Experimental Animal Co., Ltd., and the mice were raised under conditions of a temperature of 21 to 23 °C and a humidity of 40 to 60%. Bovine type II collagen (Chondrex, WA, USA) was dissolved in 0.1 N acetic acid at a concentration of 4 mg/mL, and then bovine type II collagen was mixed with an equal amount of complete Freund's adjuvant (Chondrex, WA, USA). A homogeneous suspension was prepared using a T-connector connected to a 3 mL syringe while cooling on ice. After confirming that the suspension was well prepared, the mouse's tail root was sterilized with an alcohol swab, and 100 μg of collagen was injected into the skin of the tail root. After 2 weeks, 100 μg of bovine type II collagen was dissolved, mixed with incomplete Feund's adjuvant (Difco, MI, USA) at a ratio of 1:1, and then administered to the soles of the feet.

3-2. Administration of Samchil Sukjihwang Complex Extract (Preparation Example 1-1) of the present invention

From the 4th day after the second administration of bovine type 2 collagen, the complex extracts of Samchil and Sukjihwang prepared in Preparation Example 1-1 were dissolved in water to make 30 mg/mL and 60 mg/mL, and a 1 mL syringe once daily 5 mL/kg was administered directly into the mouth of the mouse by connecting the zone to the zone.

3-3 Observation of pathological progress of arthritis: Visual observation and measurement

Visual observation of arthritic lesions was performed using the following scores based on references.

0: No swelling and swelling, 1: Slight redness and swelling of the joint, 2: Clear redness and swelling of the joints, 3: Severe degree of redness and swelling, including the interphalangeal joint, 4: Swelling throughout the entire joint this extreme.

Therefore, the highest score for joint lesions was 16 for the left and right anterior and hind limbs per mouse, and 4 was the highest score for each leg (Courtenay JS, Dallman MJ, Dayan AD, et al.: Immunisation against heterologous type II collagen induces arthritis in mice. Nature 283: 666-668. 1980).

As shown in Figure 3, when the composite extract (Preparation Example 1-1) of the present invention was orally administered to a mouse suffering from type 2 collagen-induced arthritis, the pathological progression of arthritis from the 6th week compared to the control group. was markedly suppressed.

Example 4. Confirmation of periodontitis treatment effect in periodontitis model

Periodontal disease is a chronic disease with multiple causes. One of the most important causes of periodontal disease is the action of bacteria. On the surface of teeth, there is always a bacterial film formed by proteins and food in saliva, to which various bacteria in the oral cavity are attached and active. Most of the periodontal patients have defects in the function of the body's defense cells, so they cannot resist various products produced by these bacteria and cause severe tissue destruction.

The experimental model of this embodiment binds silk sutures and wires to the teeth of beagle dogs so that plaque and calculus, which are the main causes of periodontal disease, naturally occur, resulting in inflammation and consequent alveolar bone resorption was created according to the principle of Preparation Example to examine the effect of the extract according to the present invention after inducing periodontal disease for 8 weeks by ligating molars (molars) of beagle dogs with silk wire (6 teeth per dog) and supplying liquid food to induce periodontal disease and releasing the ligation. 1-1 Tablets containing 37.5 mg, 150 mg, and 450 mg were administered to dogs weighing about 10 kg (three dogs per group), one tablet twice a day. To 3 animals in the negative control group, the same tablet prepared as an excipient was administered with one tablet twice a day. One normal group was not administered. Clinical attachment level (CAL), a clinical index, was measured before drug administration and after drug administration for 1 week. The results are shown in FIG. 4 . 1 is a normal control group, 2 is a negative control group, 3 is Preparation Example 1-1 7.5 mg/kg administration group, 4 is Preparation Example 1-1 30 mg/kg administration group, 5 is Preparation Example 1-1 90 mg/kg administration group, respectively indicate

As shown in Figure 4, after inducing periodontal disease, the CAL of the negative control group was 1.62 mm, whereas the CAL of the group administered with the composite extract of Samchil and Sukjihwang of Preparation Example 1-1 showed a concentration-dependent decrease, 30, 90 In the case of the mg/kg administration group, it decreased to 1.17 mm and 1.00 mm, respectively.

Example 5. Confirmation of hyperalgesia by heat stimulation (hot plate method)

The mouse was placed on a hot pad maintained at 30 °C and allowed to acclimatize for about 10 minutes. The mouse was placed on a hot plate maintained at a constant temperature (55 ± 1 ° C) and the time to lick the front or hind paws or jump off (hot plate latency) was measured (Jones CK, Peters SC, Shannon HE. Efficacy of duloxetine, a potent and balanced serotonergic and noradrenergic reuptake inhibitor, in inflammatory and acute pain models in rodents. J Pharmacol Exp Ther. 2005, 312(2):726-732.). Measurement was performed within 30 seconds (cut-off time: 30 seconds) to prevent tissue damage, excluding the stepping off of the mouse to move during measurement. Test substance or vehicle (0.5% CMC) was administered before and after 60 minutes and 120 minutes after administration, but only animals that showed a reaction within 8 seconds before administration were selected and used. In the case of mice that did not respond within the time limit (30 seconds) when measuring the avoidance response after 60 and 120 minutes, the cut-off time (30 seconds) was recorded (Choi JH, Cha DS, Jeon H. Anti-inflammatory and anti-nociceptive properties of Prunus padus. J Ethnopharmacol. 2012, 21;144(2):379-386). The analgesic effect result is calculated as the hot plate latency time (sec) before administration (baseline) and the avoidance reaction time after 120 minutes (120 min) (Patel RB1, Pawar VD, Prajapati KD, Sonara BM, Deshpande SS, Shah GB, Jain MR. Anti-nociceptive and anti-allodynic activity of aliskiren in various pain models. Eur J Pharmacol. 2013, 15;708(1-3):80-87), the result of MPE (%) is MPE ( %) (percentage of maximal possible effect) = [(post drug latency - predrug latency) / (cut off time - predrug latency)] × It was calculated using a formula of 100 (Demir Ozkay U1, Can OD. Anti-nociceptive effect of vitexin mediated by the opioid system in mice. Pharmacol Biochem Behav. 2013, 109:23-30). The results are shown in FIG. 5 .

As shown in Figure 5, when the composite extract of Samchil and Sukjihwang of Preparation Example 1-1 was administered at 25, 100, and 300 mg/kg, the MPE (%) was 14.3%, 15.2%, and 18.4%, compared to 2.45% in the control group. , and the group administered with 300 mg/kg of the composite extract of Samchil and Sukjihwang of Preparation Example 1-1 showed efficacy similar to that of celecoxib 50 mg/kg.

Example 6. Confirmation of pain treatment effect through tail avoidance test (tail-flick method)

An area approximately 3 cm from the end of the tail of the mouse was measured to mark the location to stimulate the heat source, and a mark was made on the side where light does not reach so as not to affect the response (Patel RB1, Pawar VD, Prajapati KD, Sonara BM, Deshpande SS, Shah GB, Jain MR. Anti-nociceptive and anti-allodynic activity of aliskiren in various pain models. Eur J Pharmacol. 2013, 15;708(1-3):80-87). An analgesiometer (Ugo Basile, model No. 37360, Varese, Italy) is used to measure the noxious heat tail avoidance response, and the time required from applying a heat source to the mouse's marked tail until it shows an avoidance response is measured with a stopwatch. (Ben-Bassat J, Peretz E, Sulman FG. Analgesimetry and ranking of analgesic drugs by the receptacle method. Arch Int Pharmacodyn Ther. 1959;122:434-47). Since the tail tissue of rats is damaged if noxious thermal stimulation continues, the exposure time to noxious thermal stimulation was limited to 10 seconds (cut-off time) for tissue protection (Choi JH, Cha DS, Jeon H. Anti-inflammatory and anti-nociceptive properties of Prunus padus. J Ethnopharmacol. 2012, 21;144(2):379-386). The frequency of measurement was performed before administration of the test substance or vehicle (0.5% CMC) and after 30 minutes, 60 minutes, and 120 minutes after administration. In the case of mice that did not respond within the time limit when measuring the avoidance response, the cut-off time (10 seconds) was written (Choi JH, Cha DS, Jeon H. Anti-inflammatory and anti-nociceptive properties of Prunus padus. J Ethnopharmacol. 2012 , 21;144(2):379-386). The result was calculated as tail flick latency time (sec), avoidance reaction time of 0 (baseline), 30 minutes (30 min), 60 minutes (60 min), and 120 minutes (120 min).

As shown in Figure 6, when the composite extract of Samchil and Sukjihwang of Preparation Example 1-1 was administered at 50, 200, and 600 mg/kg, the MPE (%) was 4.7%, 10.7%, and 16.0%, respectively, compared to 1.63% of the control group. It increased in a concentration-dependent manner, and the 600 mg/kg administration group showed similar efficacy to celecoxib 100 mg/kg.

Example 7. Confirmation of analgesic effect by acetic acid-induced writhing test

The analgesic effect was measured according to the method of Koster and Demir Ozkay (Koster, R., Anderson, M., Beer, E.J., 1959. Acetic acid for analgesic screening. Federation Proceeds 18, 412-416, Demir Ozkay U, Ozkay Y, Can OD. Synthesis and analgesic effects of 2-(2-carboxyphenylsulfanyl)-N-(4-substitutedphenyl)acetamide derivatives. Med Chem Res. 2011;20:152-157). A test substance or vehicle (0.5% CMC) was administered by dose to mice fasted for 12 hours 60 minutes before administration of acetic acid. 60 minutes after administration, intraperitoneal injection of 0.6% acetic acid-physiological saline (1% in 0.9% sterile saline) at a dose of 0.1 mL/10 g b.wt. (body weight) (10 mL/kg) was performed in individual cages. The number of occurrences of writhing was measured for 10 minutes in individual cages from 5 minutes after injection (65 minutes after test substance administration). As a result, the total number of writhes (writhes/10 min) is calculated as the total number of times during the observation time, and the pain inhibition rate, inhibition % = [(control mean - treated mean) / control mean (control) mean)] × 100 (Choi JH, Cha DS, Jeon H. Anti-inflammatory and anti-nociceptive properties of Prunus padus. J Ethnopharmacol. 2012, Nov 21;144(2):379-386 Demir Ozkay U1, Can OD. Anti-nociceptive effect of vitexin mediated by the opioid system in mice. Pharmacol Biochem Behav. 2013; 109:23-30).

As shown in Figure 7, it was confirmed that Writhing syndrome caused by administration of acetic acid was inhibited by 33.3%, 51.3%, and 61.5%, respectively, when the complex extract of Samchil and Sukjihwang of Preparation Example 1-1 was administered at 50, 200, and 600 mg/kg. This was a statistically significant result compared to the control group.

Example 8. Confirmation of analgesic effect by Rotarod test

The Rotarod method is to evaluate the motor coordination of animals and was measured using the mouse rotarod system (Panlab, Barcelona, Spain) (Bastos LF1, Prazeres JD, Godin AM, Menezes RR, Soares DG, Ferreira WC, Dutra MM, Machado RR, Coelho MM. Sex-independent suppression of experimental inflammatory pain by minocycline in two mouse strains. Neurosci Lett. 2013 Oct 11;553:110-4). The rotational speed of the Rotarod was measured by setting it to 7 rpm for mice and 10 rpm for rats before and after test substance administration to mice and rats, respectively. Before administration of the test substance, the baseline value was obtained by placing the mouse or rat on the rotating bar and checking the time when it fell. At this time, the cut-off time was set to 1 minute. After calculating the baseline value, the animals were administered a test substance or vehicle (0.5% CMC) according to group assignment. The test was performed 120 minutes after administration of the test substance.

As shown in Figure 8, it was confirmed that the composite extract of Samchil and Sukjihwang of Preparation Example 1-1 did not affect motility.

Through the above Examples 1 to 8, it was confirmed that the composition of the present invention has excellent preventive and/or therapeutic effects on inflammatory diseases (eg, arthritis (osteoarthritis, rheumatoid arthritis), periodontitis) or pain.

Claims (15)

A pharmaceutical composition for preventing or treating inflammatory diseases or pain, wherein the weight ratio of ginsenoside Rg3 to ginsenoside Rb1 is 0.20 to 25, and contains a complex extract of Samchil and Sukjihwang as active ingredients. delete The pharmaceutical composition according to claim 1, wherein the complex extract is extracted from a mixture of Samchil and Sukjihwang. The pharmaceutical composition according to claim 3, wherein the weight ratio of Samchil and Sukjihwang contained in the mixture is 3:1 to 18:1. According to claim 1, wherein the composite extract is a pharmaceutical composition in the form of a mixture of a single extract of Samchil and a single extract of Sukjihwang. [Claim 6] The pharmaceutical composition according to claim 5, wherein the weight ratio of the single extract of Samchil and the single extract of Sukjihwang is 1.5: 1 to 9: 1. The pharmaceutical composition according to claim 1, wherein the composite extract further contains ginsenosides Rd, Rh1, Rh4, Rk1, Rk3 and Rg5 in addition to ginsenosides Rb1 and Rg3. The pharmaceutical composition according to claim 7, wherein the weight ratio of the total amount of ginsenosides Rd, Rh1, Rh4, Rk1, Rk3 and Rg5 to ginsenoside Rb1 is 1.1 to 75. The pharmaceutical composition according to claim 7, wherein ginsenoside Rg3 is included in an amount of 7.0% by weight or more of the total amount of ginsenosides Rb1, Rg3, Rd, Rh1, Rh4, Rk1, Rk3 and Rg5. The pharmaceutical composition according to claim 1, wherein the inflammatory disease is arthritis or periodontal disease. The pharmaceutical composition according to claim 10, wherein the arthritis is degenerative arthritis or rheumatoid arthritis. The pharmaceutical composition according to claim 10, wherein the periodontal disease is gingivitis or periodontitis. [Claim 13] The method of claim 12, wherein the composition is an oral composition for inflammatory diseases or pain prevention or treatment pharmaceutical composition. [Claim 14] The method of claim 13, wherein the composition for oral cavity toothpaste, mouthwash and mouthwash, and at least one selected from the group consisting of inflammatory diseases or pain prevention or treatment pharmaceutical composition. The weight ratio of ginsenoside Rg3 to ginsenoside Rb1 is 0.20 to 25, and a health functional food for preventing or alleviating inflammatory diseases or pain, containing complex extracts of Samchil and Sukjihwang as active ingredients.

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