KR100586809B1 - food supplement preventing arthritis comprising red ginseng extract - Google Patents

Abstract

The present invention relates to a red ginseng extract having anti-inflammatory activity, glucosamine, shark cartilage powder, vitamin C and MnCl ₂ , and a therapeutic food for arthritis prevention and improvement. The active ingredient included in the red ginseng extract and the therapeutic agent and health food of the present invention may be useful for preventing or treating arthritis due to its excellent anti-inflammatory, anti-arthritis, anti-pain and immunostimulating effect.

Description

Mixed preparation containing red ginseng having anti-inflammatory activity, health foods for preventing arthritis using the same as an active ingredient {food supplement preventing arthritis comprising red ginseng extract}

Figure 1a is a graph showing the results of capillary permeability after administration of red ginseng extract, glucosamine, shark cartilage powder, Willowak, Cordyceps sinensis in acetic acid-induced mice.

Figure 1b is a graph showing the results of capillary permeability measurement after administration of the mixed formulations S1 and S2 prepared in the present invention to acetic acid-induced mice.

Figure 2a is a graph showing the effect of inhibiting the edema after administration of red ginseng water extract, glucosamine, shark cartilage powder, willowak, Cordyceps sinensis in rats induced edema with carrageenan.

Figure 2b is a graph showing the edema inhibitory effect after the administration of the mixed formulations S3 and S4 prepared in the present invention to the rat induced edema with carrageenan.

Figure 2c is a graph showing the effect of inhibiting the edema after administration of the mixed formulation RGM prepared in the present invention to rats induced edema with carrageenan.

Figure 3 is a graph showing the adjuvant induced arthritis inhibitory effect by the mixed formulation RGM prepared in the present invention.

Figure 4 is a graph showing the sustained effect of weight loading stream by the mixed formulation RGM prepared in the present invention.

5 is a graph showing the analgesic inhibitory effect by the mixed formulation RGM prepared in the present invention.

Figure 6 is a graph showing the immune enhancing effect by the mixed formulation RGM prepared in the present invention.

Figure 7 is a graph showing the production of IL-6 and interferon-γ in arthritis inhibited rats by the mixed formulation RGM prepared in the present invention.

Figure 8a is a graph confirming the inhibition of nitric oxide production in mouse macrophage treated with red ginseng extract of the present invention.

Figure 8b is a Western blot picture confirming the iNOS protein production inhibitory ability in the macrophage cells treated with the red ginseng extract of the present invention.

Figure 9 is a microscopic view of histopathologically observed joint sites in arthritis inhibited rats by the mixed formulation RGM prepared in the present invention.

The present invention relates to an arthritis treatment agent and a health food for preventing and improving arthritis, and more particularly, a preparation for arthritis prevention and treatment, which is prepared by further mixing other active ingredients with a natural product having an arthritis inhibitory effect, and as an active ingredient It relates to a therapeutic agent for arthritis and a health food for preventing and improving arthritis.

Medically, arthritis is an inflammatory change in the joints that is confined to the joints, the joints are swollen, and the range of motion of the joints decreases. In particular, degenerative joint disease is osteoarthritis, the most commonly known arthritis and is often inflammatory with age. It is known that cartilage is lost without alteration and joint deforms and local degenerative change occurs. Cartilage is changed and destroyed by various biological factors. Osteoarthritis includes osteoarthritis, rheumatoid arthritis and polyarthritis. Osteoarthritis is the most common cause of chronic inability after middle age due to pain and stiffness, and the underlying pathological process is degenerative, unlike inflammatory rheumatoid arthritis (Govan, ADT et al ., Pathology Illustrated 3rd ed., Pp 811-821 , Churchill Livingstone, 1991).

In general, rheumatoid refers to chronic rheumatoid arthritis, which is known to be a refractory disease that is difficult to identify the cause of the disease because it is a disease of connective tissue distributed throughout the body centered on the joint. The incidence of chronic rheumatoid arthritis does not differ by race and geographic location, but women have a higher incidence than men, especially between 35 and 45 years of age, but the difference between men and women decreases with age. Active chronic inflammation of the synovial membrane causes stiffness in the hands and feet, pain, swelling, and degeneration in the joints and subcutaneous nodules in the knees and fingers (Kim Chang-jong, Pathophysiology, Hallym Co., pp. 664-667, 1991). The main cause of rheumatoid arthritis is not clear, but various infection theories, vitamin deficiencies, hormonal mismatches and immunological reactions have been raised (American College of Rhematology AD HOC Committee on Clinical Guidelines, 39 (5), 713). -722, 1996).

Changes in connective tissue in rheumatoid joints begin with damage to capillaries and proliferation of synovial cells, which infiltrate lymphocytes into the connective tissue, resulting in fibroblast and macrophage. Increase in lymphocytes and monocytes and the cellular immune response is strong. Various factors secreted by lymphocytes and monocytes induce polymorphonuclear neutrophils and further activate monocytes and fibroblasts to intensify the inflammatory response. Activated monocytes increase phagocytosis and secrete prostaglandin, a inflammatory mediator, plasminogen activator, collagenase, and lysosomal enzyme. Destroys tissue at the joints. The arthritis-producing cytokines, IL-1 (interleukin-1), TNF-α, granulocyte / macrophage colony stimulating factor (GM-CSF), IL-6, etc. It is produced in fibroblasts and is involved in triggering immune responses.

After being diagnosed with rheumatic disease, arthralgia and myalgia appear as early symptoms. Anti-inflammatory and analgesic anti-inflammatory drugs, NSAIDs, are the first choice for the treatment of these early symptoms.Anti-rheumatic or immunosuppressive agents (cyclophospamide, azathioprine, 6-mercaptopurine, chlorambucil, methotrexate) (Kho Ho-yeon, Korean J. Med ., 51, 271-279, 1996).

Drug therapy for the treatment of arthritis has generally been used aspirin, nonsteroidal anti-inflammatory drugs, but the reality is that side effects and fundamental problems have not been solved. In addition, aspirin, naproxine, and the like, which have been used to treat arthritis so far, have side effects such as stomach cramps and headaches, and suppress the symptoms, but have a disadvantage that eventually worsens the disease.

Panax ginseng CA Meyer ( Panax schinseng Nees) is a root belonging to the Araliaceae family and has been used as an adjunct to the treatment of various diseases in Asian countries for thousands of years. In addition, administration of ginseng saponins or extracts to chronically inflammatory animals reduces the mRNA levels of IL-1β or IL-6 (Yu and Li, Acta Pharmacol. Sin . 21, 915-918, 2000) and modulates immune function. Have been reported (Sonoda et al ., Immunopharmacology , 38, 27-294, 1998; Cabral de Oliveira et al ., Comparative Biochemistry and Physiology Part C , 130, 369-377, 2001). However, in the early 1990s, anti-inflammatory studies of ginsenoside Ro showed that ginsenoside Ro inhibited acetic acid-induced vascular permeability and was induced by compound 48/80 or carragenan. Acute edema has been reduced, but it has been reported that rats in the arthritis model did not reduce edema (Matsuda et al ., Planta Med , 56 (1), 19-23, 1990).

Red ginseng (Red Ginseng) is a ginseng that has been steamed for 6 years and dried to be less than 14% of water, and the browning reaction is promoted during the manufacturing process. Red ginseng prevents the contamination of bacteria, mold and microorganisms by reducing moisture through processing and drying, and it is easy to store and transport by reducing volume and weight. Saponin quantitative method and quality control, which is the representative active ingredient of ginseng, prevented the degradation of saponin during the processing process, and saponin, maltol, ginsenoside Rh2 and other active ingredients were additionally produced. To date, numerous studies have been conducted at home and abroad on the ingredients and pharmacological effects of red ginseng, especially red ginseng's antioxidant activity (Kwon Yong-hoon, Kim Kyung-hyun, Chun, Seong Geum-su, Jang Jae-cheol, Korea Ginseng Journal, 24 (1), 29-34, 2000).

Thus, the present inventors confirmed that the extract extracted from red ginseng had anti-inflammatory activity while searching for natural substances as an alternative to solve the side effects of the conventional arthritis treatment agent, and in addition, glucosamine, shark cartilage powder, vitamin C and MnCl ₂ The present invention was completed by confirming that the mixed preparation prepared by mixing the anti-inflammatory activity, anti-arthritis, immune strengthening, analgesic effect and the ability to inhibit nitric oxide production.

An object of the present invention is a mixed preparation for the treatment and prevention of arthritis, which contains one or more components selected from the group consisting of red ginseng extract, glucosamine, shark cartilage powder, vitamin C and MnCl ₂ having anti-inflammatory activity, as an active ingredient It is to provide an arthritis treatment containing and a health food for preventing and improving arthritis.

In order to achieve the above object, the present invention is an arthritis treatment and arthritis prevention and improvement containing one or more components selected from the group consisting of red ginseng extract, glucosamine, shark cartilage powder, vitamin C and MnCl ₂ having anti-inflammatory activity as an active ingredient Provide health food for children.

Hereinafter, the present invention will be described in detail.

The present invention provides an anti-arthritis therapeutic agent containing one or more components selected from the group consisting of red ginseng extract, glucosamine, shark cartilage powder, vitamin C and MnCl ₂ having anti-inflammatory activity.

In the therapeutic agent of the present invention, the red ginseng extract is preferably extracted using a solvent selected from the group consisting of water, alcohol and aqueous alcohol solution.

In a preferred embodiment of the present invention by adding 7 times distilled water to red ginseng extracted three times for 8 hours at 90 ℃ and then lyophilized to prepare a red ginseng water extract, and extracted three times by adding 7 times 100% ethyl alcohol to red ginseng The red ginseng alcohol extract was prepared by lyophilization, and 70% ethyl alcohol was added 7 times to red ginseng to prepare a primary extract, and 50% ethyl alcohol was added 7 times to prepare a secondary extract. A tertiary extract was prepared and lyophilized to prepare an extract of red ginseng alcohol solution.

40 to 50% by weight of red ginseng extract, 20 to 30% by weight of glucosamine, 20 to 30% by weight of shark cartilage powder, 1.25 to 1.75% by weight of vitamin C, and 1.25 to 1.75% by weight of MnCl _{2 based} on the total weight of the therapeutic agent It is preferable to include. In addition, the red ginseng extract is 43.5% by weight, glucosamine is 25% by weight, shark cartilage powder is 25% by weight, vitamin C is 1.5% by weight and MnCl ₂ is 1.5% by weight based on the total weight of the therapeutic agent of the present invention. Do.

Glucosamine provides the joints with the substances they need to repair damage from osteoarthritis or injury. In particular, glucosamine is a precursor of glycosaminoglycan in proteoglycan, which is the major component of cartilage, and most other body tissues including synovial fluid, ligaments and tendons of digestive system, circulatory system, basal mucosa in joints It is a substance that makes up.

Shark cartilage is composed of ash, protein, carbohydrates, moisture, fiber and phosphorus. Shark cartilage contains a component called chondroitin sulfate, which is part of a huge protein that makes cartilage elastic. Chondroitin sulfate has been reported to relieve the pain of mild osteoarthritis and slow the degeneration of cartilage to the same extent as nonsteroidal arthritis drugs such as aspirin and ibpropene. Shark cartilage is also known to act as angiogenesis inhibitors (Lee, A. and Langer, R. Science , 221, 1185-1187, 1983).

In a preferred embodiment of the present invention using the water as a solvent to prepare a red ginseng water extract, and administered to acetic acid-induced mice, it was confirmed that capillary permeability is inhibited (see Figure 1a ), administration to carrageenan-induced mice , It was confirmed that edema is suppressed (see FIG. 2A ). In addition, during inflammatory reactions, macrophages are activated by interferon gamma to induce nitric oxide synthase to produce nitric oxide (NO) from arginine. However, the synthesis of excess nitric oxide can injure tissues and cause autoimmune diseases or chronic inflammation. Thus, it was confirmed that the red ginseng extract was treated with macrophages at concentrations of 10 μg, 50 μg and 100 μg, which inhibited the generation of nitric oxide from macrophages (see FIGS . 8A and 8B ).

Thus, glucosamine, shark cartilage powder, vitamin C, and MnCl ₂ were mixed with 43.5: 25: 25: 1.5: 1.5, respectively, to the red ginseng extract having anti-inflammatory activity, and as a result of oral administration thereof, anti-arthritis ( FIG. 2c and FIG. 3 ), analgesic effect (see FIG. 5 ), immune-related cytokine increase (see FIG. 7 ), immunopotentiation effect (see FIG. 6 ), and the combination of the above components is useful as a therapeutic agent for arthritis. It was confirmed that it can be used.

The arthritis therapeutic agent of the present invention may be prepared as a medicament by adding one or more pharmaceutically acceptable carriers together with an active ingredient having an anti-inflammatory effect. The carrier may include, but is not limited to, saline, buffered saline, water, glycerol and ethanol, and any suitable agent known in the art (Remingtons's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA) may be used. .

The formulations of the present invention can be administered orally during clinical administration and can be used in the form of general pharmaceutical formulations, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used Is prepared using.

The formulation of the present invention may be applied differently depending on the age, sex, condition, absorption of the active ingredient, inactivation rate and excretion rate of the subject, and the drug used in combination, for example, orally administered 10 to 15 g per day. However, it is not limited thereto. The invention also includes formulations of dosage units. The formulations are present in individual dosage forms, such as tablets, coated tablets, capsules, pills, suppositories, and ampoules, wherein the amount of active compound in the drug corresponds to the fraction or multiple of the individual dosage. Dosage units may contain, for example, one, two, three or four times the individual dosage, or 1/2, 1/3 or 1/4 times. The individual dosages preferably contain an amount in which the active compound is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dosage.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and liquid preparations for oral use include suspensions, solvents, emulsions, and syrups. In addition to liquids and paraffins, various excipients may be included, such as wetting sweeteners, fragrances, preservatives and the like.

In another aspect, the present invention provides a health food for preventing and improving arthritis, containing one or more components selected from the group consisting of red ginseng extract, glucosamine, shark cartilage powder, vitamin C and MnCl ₂ having anti-inflammatory activity.

When the active ingredient having an anti-inflammatory activity of the present invention is used as a food additive, each of the above-described ingredients or mixtures thereof may be added as it is or used with other foods or food ingredients, and may be appropriately used according to conventional methods. The mixed amount of the active ingredient can be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, during the preparation of food or beverages, the active ingredient having an anti-inflammatory activity of the present invention, that is, red ginseng extract 20 to 50% by weight, glucosamine 20 to 40% by weight, shark cartilage powder 20 to 20% by weight of the total health food 40% by weight, vitamin C is 2 to 7% by weight, MnCl ₂ is preferably included in 1 to 3% by weight, red ginseng extract 40 to 50% by weight, glucosamine 20 to 30% by weight based on the total weight of health food Shark cartilage powder is 20 to 30% by weight, vitamin C is more preferably 4 to 6% by weight, MnCl ₂ is more preferably contained in 1 to 2% by weight. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. It is certain.

There is no particular limitation on the kind of food. Examples of foods to which the above substances can be added include dairy products, including drinks, meat, sausages, breads, chocolates, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, beverages, alcoholic beverages. And vitamin complexes and the like, and include all of the health foods in the conventional sense.

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

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

Example 1 Preparation of a Mixture Using Red Ginseng, Glucosamine, Shark Cartilage, Willowak, Cordyceps Sinensis

The red ginseng used 6-year-old red ginseng provided by Korea Ginseng Changchang. The dried red ginseng was put in an extraction container, and water was added about 7 times as a solvent, followed by extraction at 90 ° C. for 8 hours to make a primary extract. Thereafter, the primary extract was placed in an extraction container, and then 7 times of water was added to carry out the same process as the preparation of the primary extract to prepare a secondary extract and a tertiary extract. The prepared primary, secondary and tertiary extracts were concentrated in a concentrator to prepare a red ginseng extract. Glucosamine, shark cartilage, Cordyceps sinensis and Willowak powder were provided by Yiwoo Yang Co., Ltd. Red ginseng water extract, glucosamine, shark cartilage powder, Cordyceps sinensis and Willowak water extract were mixed to prepare a mixture formulation S1, S2, S3 and S4 ( Table 1 ).

ingredient S1 (weight ratio) S2 (weight ratio) S3 (weight ratio) S4 (weight ratio) Red Ginseng Water Extract 50 75 40 50 Glucosamine 25 25 20 16.7 Shark Cartilage Powder 25 25 20 16.7 Cordyceps sinensis 12.5 12.5 10 8.3 Willowak Water Extract 12.5 12.5 10 8.3

<Example 2> Anti-inflammatory activity through acetic acid-induced mouse capillary permeability test

Inflammatory edema, capillary permeability is a symptom caused by the leakage of plasma components out of the blood vessel, the anti-inflammatory effect can be verified by the effect on the capillary permeability, the early stage of inflammation. According to the method of Whitley (Whittle, BA, Brit. J. Pharmacol , 22, 246-253, 1964), the effect of each of the formulations and components thereof prepared on acetic acid induced mouse capillary permeability was examined. Specifically, ICR male mice weighing 25-28 g received from Samta Co., Ltd. were reared for 2 weeks or more in an experimental animal breeding room and were adapted. The kennel was adjusted to 12 hours from 8 am to 8 pm by artificial lighting, and the room temperature was maintained at 24 ± 1 ℃. Eight males, weighing 32-34 g, were used as a group, and red ginseng group 1 (250 mg / kg), red ginseng group 2 (500 mg / kg), glucosamine group (240 mg / kg) and shark cartilage. Drugs were divided into powder administration group (240 mg / kg), Willowak administration group (120 mg / kg), Cordyceps sinus administration group (120 mg / kg), S1 formulation administration group (1,250 mg / kg), and S2 formulation administration group (1,500 mg / kg). Was administered orally. Thirty minutes after oral administration, 1% Evans blue solution (Sigma Chem) was injected into the tail vein by 0.1 ml. Evans blue solution can be used as an indicator of inflammatory action using the amount of pigment of Evans blue flowing into the abdominal cavity as an indicator of permeability hyperactivity. After 20 minutes, 0.1 ml / 10 g of 0.7% acetic acid-physiological saline solution, which is a base salt, was injected intraperitoneally to increase vascular permeability. After 20 minutes, the abdominal cavity was washed with 10 ml of saline solution and collected and centrifuged at 3,000 rpm for 10 minutes. The degree of vascular permeability was analyzed under the 610 nm ultraviolet absorption spectrum. As a control drug, ibuprofen (Sigma Chem) 200 mg / kg was orally administered and compared.

All experimental results of the present invention were calculated as the mean ± standard error (M ± SE). The significance test between each group used Student's t- test, and it was judged that it was significant when p value was less than 5% (* p <0.05, ** p <0.01).

As a result, there was no significant inhibitory effect in the group administered with each component for acetic acid-induced capillary permeability of the acetic acid-induced mice. In the group administered at the 240 mg / kg dose, the values of 59.5 μg and 56.7 μg per mouse were shown, respectively, indicating an inhibitory effect of 29.6% and 32.9%, respectively. In addition, as the dose of red ginseng water extract increased, capillary permeability inhibitory effect increased. The control group administered ibuprofen showed a 58.8 μg value per mouse, indicating an inhibitory effect of 30.4% ( FIG. 1A ). Therefore, it was found that red ginseng, glucosamine and shark cartilage powder had an excellent capillary permeability inhibitory effect.

In addition, administration of Formulation S1 or Formulation S2 prepared by mixing red ginseng, glucosamine, shark cartilage, Willowak, and Cordyceps sinensis showed a significant inhibitory effect on capillary permeability. In contrast, the control group that received no treatment showed a value of 86.0 μg per mouse, whereas the group administered with Formulation S2 showed a value of 71.7 μg per mouse, indicating an inhibitory effect of 16.6%, indicating a significant anti-inflammatory effect. When ibuprofen (200 mg / kg) used as a reference drug was administered, it showed a value of 51.6 μg per mouse, showing an inhibitory effect of 40% ( FIG. 1B ). In the case of the mixed preparations S1 and S2, the red ginseng content was prepared differently, and the capillary permeability of the S2 preparation with 1.5 times more red ginseng content than the S1 preparation was much suppressed, indicating that the red ginseng was due to the anti-inflammatory activity of the red ginseng. .

Example 3 Anti-inflammatory Activity Through Carrageenan-Induced Rat Edema Experiment

Carrageenan is a sulfated polygalactan obtained from algae, especially λ-carrageenan, which is characterized by acute inflammation by selective toxic effects on macrophages (Bonney, RJ, et. al., J. Exp. Med ., 148, 261, 1977). In addition, the plasmin (plasmin) is activated to promote the production of kinin (kinin). Accordingly, edema inhibition experiments were conducted in rats induced by carrageenan according to the method of Winter et al. (Winter, CA et al., Proc. Soc. Exp. Biol. Med ., 111, 544-547, 1962). Sprag-Douri male rats of body weight 170-180 g received from Samtaco were bred for 2 weeks or longer in the laboratory. The kennel was controlled by artificial lighting to adjust the lighting time from 8 am to 8 pm for 12 hours, and the room temperature was maintained at 24 ± 2 ° C. Seven 180-220 g male rats were used as a group, and edema was induced by subcutaneous injection of 0.03 ml of 1% carrageenan (Sigma Chem) into the rear sole. 30 minutes before carrageenan injection subcutaneously, red ginseng group 1 (250 mg / kg), red ginseng group 2 (500 mg / kg), glucosamine group (240 mg / kg), shark cartilage powder group (240 mg / kg) , Willowak administration group (120 mg / kg), Cordyceps sinus administration group (120 mg / kg), S3 formulation administration group (1,000 mg / kg), S4 formulation administration group (1,000 mg / kg) divided into oral administration. The volume of edema was measured on a plethysmometer (Ugo Basile, Italy) four times at 1 hour intervals after carrageenan injection, and then in increments of edema based on the volume before injection (after induction and before paw induction). The degree of edema inhibition was assayed according to the edema growth rate described in Formula 1). The control group was orally administered with ibuprofen (200 mg / kg) as a control drug.

As a result, carrageenan-induced swelling of the soles of the rats was most severe 30 minutes after carrageenan injection, and the red ginseng administration group 2 (500 mg / kg) after 30 minutes, 1 hour, 2 hours, and 3 hours, respectively, compared to the control group. 19.5%, 29.1%, 13.0%, and 20.3% showed continuous edema suppression effect. In addition, the shark cartilage powder-treated group showed 12.2%, 4.5% and 6.8% of inhibitory effect after 30 minutes, 1 hour and 2 hours, and 30.8% after 3 hours. The control group, ibuprofen, showed a 37.9% inhibitory effect after 3 hours ( FIG. 2A ). Therefore, it was found that red ginseng, glucosamine and shark cartilage powder had edema inhibitory effects, and red ginseng had a concentration-dependent edema inhibitory effect compared to red ginseng administration group 2 compared to red ginseng administration group 1. Could know.

In addition, when the mixed formulation was administered, the mixed formulation S4 showed inhibitory effects of 32.9%, 39.5%, 34.8% and 33.5% at 30 minutes, 1 hour, 2 hours and 3 hours, respectively, compared to the control group. It showed much stronger inhibitory action than formulation S3. When the control drug ibuprofen was administered at 30 minutes, 1 hour, 2 hours and 3 hours, the inhibitory effect was 58.2%, 58.0%, 37.1% and 43.5%, respectively, compared to the control group ( FIG. 2B ). Therefore, the red ginseng content of the red ginseng contained in the mixed formulation is about twice as high as the S3 formulation.

Based on the above results, red ginseng, glucosamine and shark cartilage powder were found to have anti-inflammatory effects, and among them, red ginseng had the best anti-inflammatory effect. Accordingly, the red ginseng extract, glucosamine, and shark cartilage powder, which had the anti-inflammatory effect, were treated with 43.5%, 25.0%, 25.0%, 1.5%, and 1.5% of vitamin C, which is an antioxidant, and MnCl ₂ , an effect factor for rheumatoid arthritis, respectively. Mixing to prepare a mixed formulation Red Ginseng Mixture (RGM). The effects of RGM preparation at the concentrations of 500 mg / kg and 1,000 mg / kg were analyzed on carrageenan-induced edema of rat paws.

As a result, when carrageenan was administered 3 hours after inducing edema, the control group that received nothing increased the swelling of the sole by 70%. However, when the mixed formulation RGM was administered at the 500 mg / kg and 1,000 mg / kg concentrations, the edema was reduced to 52% and 45%, respectively, compared to the control ( FIG. 2C ). Therefore, it was found that the mixed formulation RGM containing red ginseng prepared in the present invention serves to alleviate edema.

Example 4 Antiarthritis Effect by Adjuvant-Induced Rat Arthritis Experiment

According to Pearson, CM et al., J. Exp. Med ., 133, 485-509, 1961, Freund's complete Freunds adjuvant used to induce chronic inflammation is mycobacter It is composed of bacteria and other mixtures of mycobacterium tuberculosis , which causes chronic arthritis such as chronic joint rheumatism or Behcet's disease by causing delayed allergy by antigen and antibody response in vivo. In addition, rat adjuvant-induced arthritis has been recognized to take a relatively chronic course of experimental inflammation and to involve the immune system before its expression. Therefore, according to the method of Cloud et al. (Claude, VW, et al., Arthritis and Rhematism , 12, 472-482, 1969), the anti-arthritis effect by administering the agent of the present invention to the rats induced arthritis with adjuvant Experiment. Eight Sprague-Douri male rats weighing 180-220 g were included in one group, and 0.1 ml of Freund's complete adjuvant was injected subcutaneously into the right hind paw. The hind paw volume was then measured using a flowmeter on the 5th, 8th, 12th, 15th and 18th days, and the 500 mg / kg, 1,000 mg / kg and 1,500 mg / kg concentrations of the preparation RGM were measured from the 10th day. It was administered for 1 week. As a reference drug, diclofenac (Sigma Chem) at a concentration of 20 mg / kg was used.

As a result, the control drug diclofenac showed a 16.7% inhibition rate on day 18. However, when the formulation RGM was administered at a concentration of 1,500 mg / kg for 10 days, arthritis was sustained compared to the control group with 14.3% at 5 days, 15.1% at 12 days, 17.5% at 15 days, and 13.3% at 18 days. It was found to exhibit an inhibitory effect ( FIG. 3 ).

<Example 5> Anti-fatigue effect through weight load swimming test

The anti-fatigue effect when the formulation of the present invention was administered was tested by weight-load swimming test (Moriura, T et al., Biol. Pharm. Bull . 19, 62-66, 1996). Male mice weighing 28-32 g were suspended on iron bars and divided to 10 per group based on endurance. Formulation RGM was administered at a concentration of 500 mg / kg or 1,000 mg / kg 30 min once a day for 3 days, followed by a weight-load swimming test. A weight equivalent to 10.5% of body weight was attached to the 5 cm portion of the top of the tail of the mouse. Use a 25 cm deep circulating water bath divided into partitions of width × length = 15 × 13 cm, adjust the temperature of the water in the bath to 33 ± 1 ° C, and allow the mice to swim. It was. The swimming duration was defined as the time from the start of swimming until it subsides and comes back to the surface for 5 seconds. As a reference drug, α-tocopherol acetate was suspended in gum arabic (Duksan Chemical Co.) and used at a concentration of 5 g / dl.

As a result, the control group which was not administered anything lasted 588 seconds, whereas the administration of the formulation RGM at 500 mg / kg and 1,000 mg / kg showed a sustained effect of 422 seconds and 400 seconds, indicating no antifatigue effect. ( FIG. 4 ).

Example 6 Analgesic Effect

Randall, LO and Salitto, JJ, Arch. Int. Pharmacodyn. Ther ., 111, 4.9-19, 1971, which are representative examples of analgesic experiments, are narcotic analgesics in anti-inflammatory analgesics. In narcotic analgesics, normal and inflamed feet both raise the threshold, but peripheral anti-inflammatory drugs with action points only increase the pain threshold of inflamed feet. Thus, to determine whether the formulation prepared in the present invention has an analgesic effect was performed analgesic effect experiment in accordance with the method of Randall and Salito. Eight Sprague-Douri male rats weighing 180-220 g were included in one group. The analgesic effect experiment was based on increasing the sensitivity to pain of the inflammatory site and suppressing the increased sensitivity by the analgesic agent. After overnight fasting, the rats that did not respond to 300 g of pressure were first excluded using an analgesymeter (Ugo Basile, Italy). Inflammation was induced by subcutaneous injection of 0.1 ml of a 20% brewers yeast-physiological saline suspension into the paws of the rats, and after 2 hours, the mixed formulation RGM was administered at 500 mg / kg and 1,000 mg / kg concentrations, respectively. After 0, 0.5, 1, 2, 3 hours, the pain threshold (g) was measured with an analyte. The pain threshold was slowly applied to the rat's foot at a rate of 16 g / sec, which was a pain response when the rat tried to pull out the foot or twisted its body because of pain. Analgesic activity was expressed by obtaining% of control pain threshold. Ibuprofen (200 mg / kg) was used as a control drug.

As a result, the control group steadily decreased the pain threshold until 30 minutes, 1 hour, 2 hours, and 3 hours after the yeast injection, and showed no difference between doses. On the other hand, administration of the mixed formulation RGM at a concentration of 500 mg / kg showed analgesic effects of 33.3%, 63.6%, 45.2% and 70.5% at 30 minutes, 1 hour, 2 hours and 3 hours, respectively. When RGM was administered at a concentration of 1,000 mg / kg, analgesic effects of 61.3%, 47.1% and 61.1% were observed at 1 hour, 2 hours and 3 hours, respectively. In particular, when the mixed formulation RGM was administered at a concentration of 500 mg / kg, the analgesic effect was almost similar to that of the control drug ibuprofen (200 mg / kg) at 1 and 2 hours ( FIG. 5 ).

Example 7 Immune Enhancing Activity Through Carbon Removal Experiment

Among the immune cells, macrophage plays the most important role in the immune system by mediating phagocytosis, cytokine production and inflammatory responses in the presence of antigen (Marcil, A. et al., Infect. Immun ., 70 , 6319-6329, 2002). Therefore, a carbon clearance experiment was performed in which the Wagner et al. (Wagner, H., et al., Arzneimittel-forschung , 34, 659-661, 1984) partially modified to confirm the immune enhancing action . . Ten ICR male mice from 32 g to 36 g were grouped and the mixed formulation RGM was administered at 500 mg / kg and 1,000 mg / kg concentrations once daily for three days, 30 minutes prior to the carbon removal experiment. Mice were injected via a tail vein at a dose of 10 μl / g (bw) of a carbon suspension (8-fold dilution of Rotring ink in PBS containing 1% gelatin). At 3, 6, 9 and 12 minutes after intravenous injection, blood was collected from the orbital vein, and 20 μl of blood was diluted in 1.5 ml of 0.1% sodium carbonate, followed by a UV spectrophotometer (Bio-Lad). Lab) absorbance was measured at a wavelength of 660 nm. Using the measured absorbance, the log E value was calculated, and the regression coefficient ratio (RCR = RCtr / RCc) of the formulation-administered group (RCtr) of the present invention to the control group (RCc) to which nothing was administered was determined. As a reference drug, zymosan of 50 mg / kg was used.

As a result, when the mixed formulation RGM was administered at the concentrations of 500 mg / kg and 1,000 mg / kg, respectively, regression ratios were 1.13 and 1.04, respectively, and the regression number of the reference drug Zymosan was 1.15, indicating that the formulation of the present invention It can be seen that the immune enhancing effect ( Fig. 6 ).

Example 8 Cytokine Analysis

In order to investigate the anti-arthritis mechanism of the mixed preparation RGM prepared in the present invention, the cytokine of the rats subjected to the adjuvant-induced arthritis experiment was analyzed. At the third week of the adjuvant-induced arthritis experiment performed in Example 4, blood was collected from the heart of the rat. The collected blood was coagulated at room temperature, left at 4 ° C. overnight, and centrifuged at 2,000 g for 15 minutes to separate serum. Samples were stored at minus 80 ° C until cytokine analysis, and thawed immediately before the experiment to determine the amount of IL-6 (Interleukin-6) and interferon-γ (INF-γ) in the serum by Enzyme-Linked Immunosorbant Assay. Quantification by Purified IL-6 or INF- [gamma] coated antibody was diluted in PBS (pH 7.4) to 4 [mu] g / ml concentration and 100 [mu] l was added to each 96-well microplate. After 96-well plates were incubated overnight at room temperature of 20-25 ° C., the coating solution in each well was completely removed, and 200 μl of assay solution (4% BSA in PBS, pH 7.4) was added per well for 1 hour. Incubated at room temperature. Wash each well three times with washing solution (50 mM Tris, 0.2% Tween-20, pH 8), remove water completely, and 50 μl of each standard well with IL-6 or INF-γ. Added. After incubating at room temperature for 1 hour, 50 μl of a biotin-labelled detecting antibody diluted in the assay solution at a concentration of 0.6 μg / ml was added to each well, and reacted again at room temperature. After 1 hour, the solution was washed three times with the washing solution, and completely drained, and 100 μl of HRP-conjugated streptavidin diluted 1: 8,000 in the assay solution was added to each well. The plate was incubated for 30 minutes at room temperature, washed three times with the wash solution and then completely drained. Add 100 μl of TMB substrate solution, react for 30 minutes at room temperature, add 100 μl of reaction stopper (0.18 MH ₂ SO ₄ ), and measure absorbance at 450 nm to measure IL-6 or INF in serum. The amount of -γ was analyzed.

As a result, the dose-dependent cytokine production increased when the mixed formulation RGM was administered, and especially when the mixed formulation RGM was administered at 1,000 and 1,500 mg / kg concentrations, the difference was statistically significant compared to that of the arthritis control group. Is shown ( FIG. 7 ).

Example 9 Inhibition of Production of Nitric Oxide

<9-1> Cell Culture and Nitric Oxide Production Induction

In order to confirm that the mixed agent of the present invention has anti-inflammatory activity, an experiment to suppress nitric oxide production was performed. The cells used in the experiment were mouse-derived macrophage RAW 264.7 cell line (ATCC), and each was dispensed into 24 well plates at a concentration of 2 × 10 ⁵ cells / ml using DMEM medium, respectively, at 37 ° C. and 5% CO ₂ incubator. Incubated at. After culturing the cells for one day, the red ginseng extract, which was confirmed to have anti-inflammatory activity in the present invention, was treated with 10, 50, and 100 ㎍ / mL concentrations, and then treated with LPS at 1 ㎍ / mL concentration for 2 hours, followed by culturing for 22 hours. . Indomethacin (indomethacin), an inhibitor of nitric oxide production, was treated at a concentration of 100 μg / ml and used as a positive control, and only LPS was used as a negative control.

<9-2> Nitric Oxide (NO) Formation Inhibition

The amount of NO produced from mouse-derived macrophage RAW 264.7 cells was measured using Gries' reagent as a form of NO ₂ ⁻ present in the cell culture according to the manufacturer's policy. Specifically, in 100 μl of the supernatant of the cells cultured in Example <9-1>, Greis reagent [1% (w / v) sulfanilamide in 5% (v / v) phosphoric acid 100 μl of 0.1% (w / v) naphtylethylenediamine-HCl] was mixed, dispensed into a 96 well plate, and reacted for 10 minutes to measure absorbance at 540 nm.

As a result, when the raw 264.7 cells were treated with LPS alone, the amount of nitric oxide produced was as high as 440 nM, while the positive control of indomethacin and the red ginseng extract of the present invention were treated. The amount of nitric oxide produced was significantly reduced. In addition, the red ginseng extract inhibited the production of nitric oxide with increasing concentration ( FIG. 8a ).

<9-3> iNOS protein expression inhibition

In order to investigate the correlation between the inhibition of inflammatory factor (NO) and the expression of iNOS protein by red ginseng water extract, the expression of protein was examined by Western blot. Cells and controls treated with red ginseng extract in Example <9-1> were washed with D-PBS, extracted with protein in lysis buffer, and centrifuged to obtain supernatant. The protein amount of the supernatant was quantified with a protein quantification reagent (Bio-Rad) and 50 μg of protein was taken. The extracted protein was electrophoresed on 8% SDS-polyacrylamide gel and then the protein contained in the gel electrophoresed with PVDF membrane. After blocking for 2 hours with 5% whole milk powder, iNOS primary antibody was added at a ratio of 1: 1000 and incubated overnight at 4 ° C. It was then washed three times at 15 minute intervals with TTBS solution (0.1% Tween, 0.1 M Tris.HCl, pH 7.4, 0.1 M NaCl). Next, the rabbit secondary antibody and goat secondary antibody diluted in the ratio of 1: 2000 were incubated at room temperature for 2 hours. Again rinsed with TTBS solution 3 times at 15 minute intervals and then developed.

As a result, protein expression of iNOS was increased by LPS, and red ginseng water extract inhibited the expression of iNOS protein in a similar trend to NO production inhibition ( FIG. 8B ). As described above, it was found that the reduction of NO production by the red ginseng water extract, which is an active ingredient included in the mixed preparation of the present invention, was due to the regulation of iNOS protein expression, thereby having anti-inflammatory activity.

Example 10 Pathological Assay

In the adjuvant-induced arthritis experiment, the administration of the formulation of the present invention was intended to determine the pathological changes. After collecting blood at the third week of the adjuvant-induced arthritis experiment performed in Example 3, the joints of the right leg of the rat were cut out and placed in 10% formalin solution and fixed for one week. After removing the formalin solution, an 8% formic acid solution was added and replaced daily until the calcification in the tissue was removed. The decalcified joint was embedded in paraffin, cut using a microtome, mounted on the slide, and then stained with Hematoxylin & Eosin solution for 400-fold and 1,000-fold. Histopathology of the joint was observed under a microscope at magnification.

As a result, it was found that the normal group had no damage to the cartilage area and had a very dense density. In contrast, arthritis-induced group showed cartilage damage in the joint area (arrow), and cartilage density decreased. When the mixed formulation RGM prepared in the present invention was administered at concentrations of 500 mg / kg, 1,000 mg / kg and 1,500 mg / kg, the histological change was little or very low compared to normal tissues. In particular, high doses showed little difference from normal tissues. Again, there was no difference in the group to which ibuprofen was administered compared to normal tissue ( FIG. 9 ).

Preparation Example 1 Preparation of Health Food Containing Red Ginseng Mixture

The present inventors confirmed that the red ginseng extract, glucosamine, shark cartilage powder, vitamin C, and MnCl ₂ each have anti-inflammatory activity, and a mixed preparation prepared by mixing them can be usefully used to prevent arthritis. It was. Thus, a health food containing the mixed agent prepared in the present invention as an active ingredient was prepared as follows. The mixing agent used to prepare the health food of the present invention is the mixing agent RGM (Red Ginseng Mixture) prepared in the above embodiment.

<1-1> Beverage Production

522 mg of honey

Chioctosanamide 5 mg

Nicotinamide 10 mg

Riboflavin Sodium Hydrochloride 3 mg

Pyridoxine hydrochloride 2 mg

Inositol 30 mg

Orthoic acid 50 mg

Red ginseng mixture formulation 0.48-1.28 mg

200 ml of water

With the above composition and content, drinks were prepared using conventional methods.

<1-2> Preparation of Chewing Gum

Gum base 20%

Sugar 76.36-76.76%

Red Ginseng Mixture 0.24 ~ 0.64%

1% fruit flavor

Water 2%

Chewing gum was prepared using conventional methods using the above composition and content.

<1-3> Preparation of Candy

50 to 60% sugar

Starch syrup 39.26-49.66%

Red Ginseng Mixture 0.24 ~ 0.64%

Orange flavor 0.1%

Candy was prepared using conventional methods with the above composition and content.

<1-4> Preparation of Biscuits

Force Class 1 88 kg

Gravity First Class 76.4 ㎏

16.5 kg per white

2.5 kg of salt

2.7 kg of glucose

Palm shortening 40.5 kg

5.3 kg of ammo

Medium kg 0.6 kg

0.55 kg sodium bisulfite

Rice flour 5.0 kg

Vitamin B1 0.003 kg

0.003 kg of vitamin B2

Milk Flavor 0.16 ㎏

71.1 kg of water

Whole milk powder 4 kg

Substitute powder 1 kg

0.1 kg of calcium phosphate

Spray salt 1 kg

25 kg of spray oil

Red ginseng mixture preparation 0.2-0.5 kg

Biscuits were prepared using conventional methods with the above composition and content.

<1-5> Preparation of Ice Cream

Milkfat 10.0%

Non-fat solids 10.8%

Sugar 12.0%

Starch syrup 3.0%

Emulsifying stabilizer (span) 0.5%

Spices (Strawberries) 0.15%

Water 63.31-62.91%

Red Ginseng Mixture 0.24 ~ 0.64%

Ice cream was prepared using conventional methods using the above composition and content.

<1-6> Preparation of Chocolate

Sugar 34.36-34.76%

Cocoa Butter 34%

Cocoa Mass 15%

Cocoa Powder 15%

Lecithin 0.5%

0.5% vanilla

Red Ginseng Mixture 0.24 ~ 0.64%

With the above composition and content, chocolate was prepared using conventional methods.

Preparation Example 2 Preparation of an Arthritis Treatment Containing a Red Ginseng Mixture

The present inventors confirmed that the red ginseng extract has anti-inflammatory activity through the above examples, which can be usefully used to prevent arthritis. Thus, a therapeutic agent containing the red ginseng extract and anti-inflammatory activity components extracted in the present invention as an active ingredient was prepared as follows. In addition, the preparation of the following therapeutic agent can be used for the application of the health food as well as the therapeutic agent.

<2-1> Preparation of Soft Gelatin Capsules

Red ginseng concentrate powder (red ginseng component 130 mg / g or more) 13.5%

Glucosamine 22.0%

Shark Cartilage Extract Powder 7.0%

Vitamin C 2.5%

Vitamin D ₃ 0.0005%

Manganese Sulfate 0.05%

5% wax

Palm oil 15%

Safflower seed oil 34.9495%

<2-2> Preparation of Tables

Red ginseng concentrate powder (red ginseng component 130 mg / g or more) 29.4%

Glucosamine 35.0%

Shark Cartilage Extract Powder 10.0%

Vitamin C 5.8%

Vitamin D ₃ 0.0005%

Manganese Sulfate 0.05%

Crystalline cellulose 10.0%

Lactose 9.0495%

Magnesium Stearate 0.7%

As described above, the red ginseng mixture formulation prepared by mixing red ginseng extract, glucosamine, shark cartilage powder, vitamin C, and MnCl ₂ is excellent in inhibiting arthritis, and thus may be useful for preparing arthritis treatment and arthritis prevention and improvement health food. Can be.

Claims (4)

A health food for preventing and improving arthritis, comprising 40 to 50% by weight of red ginseng extract, 20 to 30% by weight of glucosamine, and 20 to 30% by weight of shark cartilage powder based on the total weight of the therapeutic agent. According to claim 1, Red ginseng extract is arthritis prevention and improvement health food, characterized in that the extract by water, alcohol, or a mixture thereof. 2. The health food for preventing and improving arthritis according to claim 1, further comprising 1.25 to 1.75 wt% of vitamin C and 1.25 to 1.75 wt% of MnCl ₂ . delete

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