KR20220073225A - Method for preparing mixture of Scutellariae Radix extract and Coptidis Rhizoma extract having excellent improvement and treatment for rheumatoid arthritis, and composition for preventing or treating rheumatoid arthritis comprising thereof - Google Patents

Abstract

The present invention relates to a method for preparing a mixture of golden extract and yellow lotus extract, which has excellent efficacy in improving and treating rheumatoid arthritis, and to a composition for preventing or treating rheumatoid arthritis, including the mixture A method for preparing a mixture of a golden extract and an extract of rhododendron having an effect of improving or treating rheumatoid arthritis, comprising the steps of preparing each and mixing the golden extract and the golden rhododendron extract in a weight ratio of 1:1, the golden extract prepared by the method And it relates to a pharmaceutical composition and health functional food for the prevention or treatment of rheumatoid arthritis comprising a mixture of yellow lily extract as an active ingredient. The mixture of golden extract and yellow liana extract prepared by the method of the present invention inhibits the proliferation and migration of synovial cells, inhibits the expression or production of inflammatory cytokines IL-6, IL-1β, TNF-α, CCL2 and CXCL2, Effectively prevents, improves and treats rheumatoid arthritis because it has all of the activity of inhibiting infiltration of immune cells, inhibiting pannus formation, inhibiting cartilage destruction, and inhibiting bone erosion, and has no side effects as a mixture of natural herbal extracts It can be usefully used in medicines and health functional foods for

Description

A method for preparing a mixture of golden extract and yellow lily extract having excellent improvement and treatment efficacy of rheumatoid arthritis, and a composition for preventing or treating rheumatoid arthritis comprising the mixture for rheumatoid arthritis, and composition for preventing or treating rheumatoid arthritis comprising thereof}

The present invention relates to a method for preparing a mixture of Scutellariae Radix extract and Coptidis Rhizoma extract, which is excellent in improving and treating rheumatoid arthritis, and to a composition for preventing or treating rheumatoid arthritis, comprising the mixture .

Rheumatoid arthritis (RA) is an autoimmune disease in which genetic factors, environmental factors, immunological factors, etc. are interrelated and cause various clinical features. In particular, it can be said to be a complex genetic disease involving several genes rather than one.

Rheumatoid arthritis is a chronic systemic inflammatory disease that affects the joints and is pathologically characterized by thickening of the synovial membrane and infiltration of inflammatory cells and destruction of the joint due to this. At first, it is known that T cells are activated by the immune response, which initiates an inflammatory response, but as the disease progresses, the influence of T cells decreases and cartilage destruction occurs due to abnormal proliferation and activation of synoviocytes. Due to the activation of degrading enzymes and bone-destroying factors, joint destruction occurs continuously. Cells present in the synovial membrane are very diverse, including lymphocytes, macrophages, monocytes, fibroblasts, neutrophils, NK cells, and mast cells. The development of rheumatoid arthritis is complicated by the interaction of these various cells with pro-inflammatory cytokines (TNF-α, IL-1, IL-6, IL-17, etc.), which is known to be related to various symptoms of rheumatoid arthritis. have.

Symptoms of rheumatoid arthritis include inflammation, infiltration of immune cells, and generation of new blood vessels in the synovial membrane of peripheral joints such as fingers, toes, and wrists. and deformation appear. In addition to joint symptoms, organ involvement (lung, kidney, heart), osteoporosis, vasculitis, etc. may also accompany, and symptoms such as pain, stiffness, and swelling typically appear slowly over several weeks in several joints. In about two-thirds of patients, fatigue, anorexia, general weakness, and vague musculoskeletal symptoms precede, followed by synovitis.

On the other hand, as a therapeutic agent for rheumatoid arthritis, anti-inflammatory steroids (eg, prednisolone), non-steroidal anti-inflammatory drugs (eg, indomethacin, aspirin), immunosuppressants [eg, cyclosporine A, tacrolimus (FK506), methotrexate, cyclophosphamide) , azathioprine], disease-modifying antirheumatic agents (eg, gold salt preparations) are being used.

However, although anti-inflammatory drugs can control inflammation and relieve some of the symptoms, it is difficult to stop the progression itself, and non-steroidal anti-inflammatory drugs cannot prevent joint cartilage loss or disease progression, and when used for a long period of time, gastroduodenal ulcer Gastrointestinal side effects such as Steroid hormones are drugs that control inflammation, but they have side effects that can cause round faces, weight gain, diabetes, and high blood pressure.

Therefore, most of the drug treatments for rheumatoid arthritis developed so far are used only for the purpose of reducing pain, but there is no drug or surgery method that gives a fundamental therapeutic effect. Therefore, there is a need to develop a new therapeutic agent for rheumatoid arthritis that has excellent preventive and therapeutic effects without causing side effects.

Accordingly, the present inventors were developing a new therapeutic agent that can effectively treat rheumatoid arthritis without side effects using natural herbal medicines, and established optimal conditions for preparing a mixture with excellent rheumatoid arthritis treatment effects using gold and yellow lilies. The present invention was completed by confirming that the prepared mixture of gold and yellow lily extract can effectively improve and treat rheumatoid arthritis without side effects.

Republic of Korea Patent Publication 10-2011-0016825

Accordingly, it is an object of the present invention to provide a method for preparing a mixture of a golden extract and an extract of H.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of rheumatoid arthritis comprising a mixture of golden extract and yellow liana extract prepared by the method of the present invention as an active ingredient.

Another object of the present invention is to provide a health functional food for the prevention or improvement of rheumatoid arthritis comprising a mixture of golden extract and yellow liana extract prepared by the method of the present invention as an active ingredient.

In order to achieve the object of the present invention, the present invention comprises the steps of preparing a silver gold extract and a hwangryeon extract, respectively; And it provides a method for preparing a mixture of the golden extract and the golden lily extract having an effect of improving or treating rheumatoid arthritis, comprising the step of mixing the golden extract and the rhododendron extract in a weight ratio of 1:1.

In one embodiment of the present invention, the golden extract or hwangryeon extract may be a water extract obtained using purified water.

In one embodiment of the present invention, the golden extract or extract obtained by adding purified water 10 to 12 times the weight of dried gold or yellow lily and heating at a temperature of 100 to 120 ° C. for 2 to 4 hours. After filtration, it may be concentrated under reduced pressure at 50 to 60° C. to obtain a golden extract or an extract of Hwangryeon.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of rheumatoid arthritis, comprising a mixture of golden extract and hwangryeon extract prepared by the method of the present invention as an active ingredient.

In addition, the present invention provides a health functional food for the prevention or improvement of rheumatoid arthritis, comprising a mixture of golden extract and yellow liana extract prepared by the method of the present invention as an active ingredient.

In one embodiment of the present invention, the mixture of the golden extract and Hwangryun extract, inhibits proliferation and migration of synovial cell lines; inhibition of the expression or production of the inflammatory cytokines IL-6, IL-1β, TNF-α, CCL2 and CXCL2; inhibition of infiltration of immune cells into joint sites; inhibition of pannus formation; and inhibition of cartilage destruction and bone erosion; It may be one with activity.

The mixture of golden extract and yellow liana extract prepared by the method of the present invention inhibits proliferation and migration of synovial cells, inhibits the expression or production of inflammatory cytokines IL-6, IL-1β, TNF-α, CCL2 and CXCL2, joint region It has all of the activity of inhibiting the infiltration of immune cells into the liver, inhibiting the formation of pannus, inhibiting cartilage destruction, and inhibiting bone erosion. It can be usefully used in medicines and health functional foods for

1 shows the results of analyzing the synovial cell proliferation inhibitory activity of a mixture of extracts and extracts prepared under different formulations and extraction conditions using gold and yellow lily in an embodiment of the present invention.
2 is a synovial cell line fluorescently stained on a plate dedicated for wound healing assay, cultured until a confluent layer is formed, and then scratches are formed. After each concentration of the mixed extract prepared under the mixing and extraction conditions and the mixture of extracts were treated, the cell density in the scratch formation area was measured to analyze the activity of inhibiting movement of synovial cells.
3 is a 96-well plate after dispensing a synovial cell line, pre-treated with a mixture of gold and yellow lotus extracts prepared under the conditions of the formulation ratio 4 of the present invention for 1 hour, followed by culturing by additional treatment with LPS and PMA, and then cells The results of analyzing the RNA levels of IL-6, IL-1β, CCL2 and CXCL2 genes in the lysate are shown.
Figure 4 is after administration of methotrexate (MTX) and a mixture of gold and yellow lily extract prepared under the conditions of the compounding ratio 4 of the present invention to rheumatoid arthritis-induced mice, respectively, clinical indicators for rheumatoid arthritis activity over time ( CAI) and the frequency of occurrence are analyzed.
Figure 5 is after administration of methotrexate (MTX) and a mixture of gold and yellow lily extract prepared under the conditions of the compounding ratio 4 of the present invention to rheumatoid arthritis-induced mice, respectively, the degree of improvement of arthritis symptoms in the hind paws of each mouse visually Shows the results confirmed by .
Figure 6 shows the hind paws of rheumatoid arthritis mice administered with methotrexate (MTX) and a mixture of golden and yellow lily extracts prepared under the conditions of the compounding ratio 4 of the present invention, respectively, through hematoxylin and eosin staining. Shows the results of histomorphological analysis of synovial hyperplasia, pannus formation, cartilage destruction and bone erosion.
Figure 7 is matrix staining through toluene blue staining for hind paws tissues of rheumatoid arthritis mice, each administered with a mixture of methotrexate (MTX) and a mixture of gold and yellow lotus extracts prepared under the conditions of the compounding ratio 4 of the present invention ( The results of histomorphological analysis of matrix staining), surface regularity and cartilage thickness are shown.
Figure 8 shows the hind paws of rheumatoid arthritis mice, each administered with a mixture of methotrexate (MTX) and golden and yellow lotus extracts prepared under the conditions of the compounding ratio 4 of the present invention, CD3, F4/80 and ICAM. The results of analyzing the infiltration of inflammatory cells by immunohistochemical staining using the -1 antibody are shown.
FIG. 9 shows inflammatory cytokines IL-6, IL-1β and It shows the result of analyzing the transcript level of TNF-α by RT-PCR.

The present invention is characterized in that it provides a method for preparing a mixture of a golden extract and an extract of golden lily, which is excellent in improving or treating rheumatoid arthritis, by using natural herbal medicines gold and yellow lily.

The gold used in the present invention ( Scutellariae Radix ) grows in the grasses of the mountains, the height is 20 ~ 60cm, grows in several stems and leaves, hairy and branched. The leaves are opposite, lanceolate, and the edges are flat. Flowers bloom in July-August, turn purple, and run in raceme inflorescences biased to one side. The corolla is curved at the bottom, standing upright, cylindrical, 1.5-2.5cm long, and shaped like a lip. The calyx is bell-shaped. 2 out of 4 stamens are long and 1 pistil, and the ovary is deeply divided into 4 stamens. The fruit bears fruit in September and has a round shape inside the calyx. The root is conical and the flesh is yellow. Gold contains significant amounts of free-B-ring flavonoids, including baicalein, baicalin, wogonin and baicalenoside, and has been reported to have antiviral, antioxidant and anticancer effects. , in particular, it has been reported to have central nervous system effects such as sedative and anticonvulsant effects.

The yellow lily ( Coptidis Rhizoma ) is native to China and is also cultivated in Korea, Japan, China, etc. for herbal medicine. Grows in eczema soils in the shade of forests in mountainous areas. The underground stem is thick and stretches sideways, with many beard roots, 4-5 root leaves at the end of the stem, and the length is 10-27cm. The leaves are compound leaves with three small leaves, and the small leaves are slightly hard, sharp and shiny. The base of the petiole becomes the sheath and there are saw teeth on the edge of the leaf. From March to April, 2-3 white flowers bloom on a peduncle that grows to about 10 cm in diameter. There are male and female flowers. The white sepals are 5-6 long and look like petals. Petals are small, linear with 8 to 15 petals. There are many stamens, and the number of pistils is 9 to 16. The ovary stalk grows, and on top of it, the fruit (袋果) is lined up in a ring shape. The name is given because the cross section of the stem and underground stem is dark yellow.

In oriental medicine, around November, the roots of 5-6 year old yellow lily and yellow rhododendrons are collected and dried in the sun. · Vomiting, dysentery, heart disease, fever, mental instability, sore throat, hematemesis, nosebleeds, bleeding, and burns.

The present invention conducted a study to develop an optimal herbal combination drug with excellent therapeutic effect for rheumatoid arthritis using such gold and yellow lotus, and as a result, a mixture of golden extract and yellow lotus extract in a weight ratio of 1:1 was used for rheumatism. It was confirmed that the therapeutic effect of arthritis was excellent.

In one embodiment of the present invention, each herbal composition composition was prepared using gold and yellow lily under different mixing ratios and different extraction conditions, specifically, after mixing gold and yellow lily in a weight ratio of 3:1 as a composition of ratio 1 , to prepare a mixed extract by extraction, and after mixing gold and yellow lotus in a weight ratio of 1:1 as a composition of compounding ratio 2, extracted to prepare a mixed extract, as a composition of mixing ratio 3, extracts of gold and yellow lotus were prepared first, respectively, A mixture of extracts was prepared by mixing the golden extract and the yellow lotus extract in a 3:1 weight ratio, and golden and yellow lotus extracts were first prepared as a composition of a combination ratio of 4, respectively, and the golden extract and yellow lotus extract were mixed in a 1:1 weight ratio to obtain the extracts. A mixture was prepared.

In order to confirm the improvement and therapeutic effect of rheumatoid arthritis for each composition prepared in compounding ratios 1 to 4, the effect on the proliferation and migration of synovial cells from rheumatoid arthritis patients was analyzed. As a result, the compounding ratio 1 Among the compositions of 4 to 4, it was found that a mixture of a composition of a composition ratio of 4, that is, a mixture of golden extract and yellow lotus extract in a 1:1 weight ratio, exhibited the most excellent activity for inhibiting proliferation and migration of synovial cells.

Therefore, the present inventors found that even if the same type of herbal medicine was used, the pharmacological activity was different depending on the compounding ratio and extraction conditions. It was found to be most suitable for use for improvement or treatment.

In addition, there are a myriad of herbal medicines having pharmacological activity, but in the case of using complex herbal substances, the desired pharmacological activity can be confirmed only through experiments. This is because extracts of a single plant or single medicinal crop also contain numerous components, and there are still many unidentified components. Rather, the reaction may result in inhibition of pharmacological activity, and harmful side effects may occur in the body.

On the other hand, the mixture of golden extract and yellow lotus extract prepared by the method of the present invention can more effectively treat rheumatoid arthritis by interaction of active ingredients contained in gold and yellow lotus without causing the above-described problems.

Therefore, the present invention comprises the steps of preparing a golden extract and a hwangryeon extract, respectively; And it provides a method for preparing a mixture of the golden extract and the golden lily extract having an effect of improving or treating rheumatoid arthritis, comprising the step of mixing the golden extract and the rhododendron extract in a weight ratio of 1:1.

In the present invention, the process for preparing the golden extract or hwangryeon extract can be prepared according to a conventional method for obtaining an extract from a natural product known in the art, that is, using a conventional solvent under conditions of conventional temperature and pressure. can be extracted. For example, extraction may be performed using one or more solvents selected from the group consisting of water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

In addition, the method of extracting the extract using a solvent may be extracted through various methods such as hot water extraction, cold extraction, reflux extraction, and ultrasonic extraction, but is not limited thereto.

The prepared extract may then be filtered or concentrated or dried to remove the solvent, and both filtration, concentration and drying may be performed. For example, the filtration may be performed using filter paper or a reduced pressure filter, the concentration may be performed by a vacuum concentrator, and the drying may be performed by a freeze-drying method, but is not limited thereto.

Preferably, the golden extract or yellow lotus extract is filtered after filtering the extract obtained by adding purified water 10 to 12 times the weight of dried gold or yellow lotus and heating it at a temperature of 100 to 120° C. for 2 to 4 hours, then 50 to It may be concentrated under reduced pressure at 60° C. to obtain a golden extract or an extract of hwangryeon.

In one embodiment of the present invention, each extract obtained by adding purified water 10 times the weight of gold or yellow lily by weight, heating at 100° C. for 3 hours, filtering each extract obtained, and then concentrating under reduced pressure at 60° C., and then vacuum drying Thus, golden extract or hwangryeon extract was prepared, respectively.

Thereafter, the prepared golden extract and yellow lotus extract were mixed in a weight ratio of 1:1 to prepare a mixture of gold extract and yellow lotus extract having rheumatoid arthritis improvement or therapeutic effect according to the present invention.

In addition, the present invention may provide a pharmaceutical composition for the prevention or treatment of rheumatoid arthritis, comprising as an active ingredient a mixture of the golden extract and Hwangryeon extract prepared by the method of the present invention.

In one embodiment of the present invention, it was confirmed whether the composition in which the golden extract and the yellow lotus extract were mixed in a weight ratio of 1:1 had an effect on the treatment of rheumatoid arthritis. There is an inflammatory cytokine IL-6, IL-1β, TNF-α, CCL2 and CXCL2 expression or production inhibitory activity, inhibition of the infiltration of immune cells into the joint site, pannus (pannus) formation inhibition and cartilage destruction and bone erosion inhibitory activity were confirmed through experiments.

Rheumatoid arthritis is characterized by inflammation and proliferation of synovial cells. In patients with rheumatoid arthritis, synovial cells are characterized by abnormal proliferation like cancer cells. make arthritis worse. In addition, inflammatory cytokines and chemokines are related to TNF-α (tumor necrosis factor-α) and IL-1β, and in response to these inflammatory cytokines, synovial cells rapidly proliferate and pannus ( Pannus) is formed, which destroys cartilage and joint bones.

Therefore, the mixture of the golden extract of the present invention and the extract of chrysanthemum extract has excellent proliferation and migration inhibitory activity of synovial cells, and can effectively treat rheumatoid arthritis by inhibiting the expression and generation of inflammatory mediators.

In an embodiment of the present invention, after treating the synovial cells with a mixture of the golden extract of the present invention and the extract of H. rhododendron, the expression levels of inflammatory cytokines IL-6, IL-1β, TNF-α, CCL2 and CXCL2 are measured. However, it was confirmed that all of the expression levels of the inflammatory cytokines were significantly reduced by the treatment of the mixture of the present invention in the synovial cells induced by inflammation with LPS.

Furthermore, in order to confirm through animal experiments whether the mixture of the golden extract of the present invention and the golden rhododendron extract can actually treat rheumatoid arthritis, the present inventors applied a mixture of the golden extract of the present invention to collagen-induced rheumatoid arthritis mice. After administration, clinical evaluation of rheumatoid arthritis activity was performed, and the infiltration of immune cells in joint tissues was analyzed, and the effect on pannus and cartilage destruction and bone erosion was analyzed. As a result, the present invention It was shown that the activity index of rheumatoid arthritis was decreased by the mixture treatment of golden extract and yellow lotus extract, and the infiltration of immune cells was also suppressed, inhibition of pannus formation, cartilage destruction and bone erosion were all inhibited, and inflammatory cytokines Expression was also shown to be reduced in the joint tissue.

In addition, the therapeutic effect of the mixture of the golden extract of the present invention and the extract of yellow liana was found to be almost similar to that of methotrexate (MTX) used as a therapeutic agent for rheumatoid arthritis.

Therefore, the present inventors found that the mixture of the golden extract and the rhododendron extract according to the present invention can be used as a novel treatment for rheumatoid arthritis.

In the present invention, the pharmaceutical composition may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredients of the present invention, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, and expanding agents. , lubricants, lubricants or flavoring agents may be used.

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

Formulations of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectables. For example, for formulation in the form of a tablet or capsule, the active ingredient may be combined with an orally, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or required, suitable binders, lubricants, disintegrants and color-developers may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets. Furthermore, by using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA by an appropriate method in the field, it can be preferably formulated according to each disease or component.

The active ingredient of the present invention may be included in an amount of 0.1 to 30% by weight based on the total weight of the composition.

In addition, the active ingredient of the present invention may be administered in a therapeutically effective amount in combination with one or more therapeutic agents (pharmaceutical combination). For example, when the active ingredient of the present invention is used in combination with a chemotherapeutic agent, a synergistic effect may occur. When the active ingredient of the present invention is administered together with other therapies, the dosage of the co-administered compound will, of course, vary depending on the type of co-drug used, the specific drug used, the condition to be treated, and the like.

In addition, the present invention may provide a method for preventing or treating rheumatoid arthritis comprising administering the composition of the present invention to a mammal.

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

As used herein, the term "therapeutically effective amount" refers to an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human as conceived by a researcher, veterinarian, physician or other clinician, which an amount that induces amelioration of the symptoms of the disease or disorder being treated. It is apparent to those skilled in the art that the therapeutically effective dosage and frequency of administration for the active ingredient of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, and general health of the patient , sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and drugs used simultaneously may be adjusted according to various factors. In the treatment method of the present invention, in the case of adults, when the active ingredient of the present invention is administered once to several times a day, it can be administered in a dose of 0.001 mg/kg to 1000 mg/kg, but the range is particularly limited. it is not

Furthermore, since the composition according to the present invention is composed of natural herbal medicines prescribed in oriental medicine and does not cause side effects and toxicity in the body, it can be safely used even when taken for a long period of time. It can also be used as a food composition for prevention or improvement.

Therefore, the present invention can provide a health functional food for the prevention or improvement of rheumatoid arthritis, comprising a mixture of golden extract and yellow lily extract prepared by the method of the present invention as an active ingredient.

The food composition of the present invention can be easily utilized as a food effective for preventing and improving rheumatoid arthritis, for example, as a main raw material, auxiliary raw material, food additive, functional food or beverage of food.

As used herein, the term “food” means a natural product or processed product containing one or more nutrients, and preferably means a state that can be eaten directly through a certain processing process, and in a conventional sense , refers to all foods, food additives, functional foods and beverages.

Foods to which the food composition according to the present invention can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, functional foods, and the like. In addition, in the present invention, foods include special nutritional foods (eg, formula milk, infant food, etc.), processed meat products, fish meat products, tofu, jelly products, noodles (eg, ramen, noodles, etc.), breads, health supplements, seasonings Food (eg soy sauce, soybean paste, red pepper paste, mixed soy sauce, etc.), sauces, sweets (eg snacks), candy, chocolate, gum, ice cream, dairy products (eg fermented milk, cheese, etc.), other processed foods, kimchi, Pickled foods (various kimchi, pickles, etc.), beverages (eg fruit beverages, vegetable beverages, soy milk, fermented beverages, etc.), and natural seasonings (eg ramen soup, etc.) are included, but not limited thereto. The food, beverage or food additive may be prepared by a conventional manufacturing method.

In addition, the “functional food” refers to a food group or food composition that has added value to act and express the function of the food for a specific purpose using physical, biochemical, and bioengineering methods, etc. It refers to food that has been designed and processed to sufficiently express the body control functions related to and recovery, etc., and may specifically be a health functional food. The functional food may include a food supplementary additive that is pharmaceutically acceptable, and may further include an appropriate carrier, excipient and diluent commonly used in the manufacture of functional food.

Furthermore, in addition to those described above, food containing the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and fillers (cheese, chocolate, etc.), pects acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, etc., wherein the components are independently or in combination can be used by

In the food containing the food composition of the present invention, the amount of the active ingredient according to the present invention may include 0.001% to 90% by weight of the total food weight, preferably 0.1% to 40% by weight. In the case of beverage, it may be included in a ratio of 0.001 g to 2 g, preferably 0.01 g to 0.1 g, based on 100 ml, but in the case of long-term intake for health and hygiene purposes or for health control may be less than the above range, and since the active ingredient has no problem in terms of safety, it may be used in an amount greater than the above range, so it is not limited to the above range.

Hereinafter, the components and technical features of the present invention will be described in more detail through the following examples. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited by the examples.

<Example 1>

Preparation of composition having therapeutic activity for rheumatoid arthritis using herbal medicine

Compositions with excellent therapeutic activity for rheumatoid arthritis were prepared using gold and yellow lily in the following manner, and considering that pharmacological activity may be different depending on the extraction conditions even if the same herbal material is used, as shown in Table 1 below. Each composition was prepared using gold and yellow lily under different conditions. At this time, gold and yellow lilies purchased from the market were used, washed and dried.

In Table 1 below, blending ratios 1 and 2 are the weights of dried gold and yellow lotus, respectively, mixed with gold and yellow lotus at each mixing weight ratio, put into an extractor, and purified water corresponding to 10 times the weight of these mixtures is added, and then at 100 ° C. After extraction for 3 hours, a mixed extract was obtained, which was filtered, and the filtrate was concentrated under reduced pressure at 60 ° C., and then vacuum dried to prepare a mixed dry extract (mixed extract of blending ratio 1 and blending ratio 2) composition of golden and yellow lotus herbal medicines. .

In addition, in Table 1 below, mixing ratios 3 and 4 are respectively weighed and put in an extractor, respectively, dried gold and yellow lily, purified water corresponding to 10 times the weight of each herbal medicine is added, and then extracted at 100° C. for 3 hours. An extract was obtained, and after filtration, the filtrate was concentrated under reduced pressure at 60° C. and dried under vacuum to prepare dry golden extract and dried golden lily extract, respectively. Then, each of the prepared dry extracts was mixed at a mixing ratio to prepare a mixture of extracts having a mixing ratio of 3 and a mixing ratio of 4.

Preparation example (composition) mixing ratio mixed content Gold goldthread mixing ratio 1 3 One Mixed weight ratio of golden and yellow lily herbs
(Golden: After mixing yellow lotus in a 3:1 or 1:1 weight ratio,
extract to prepare a mixed extract) mixing ratio 2 One One mixing ratio 3 3 One Mixing weight ratio of golden extract and yellow lily extract
(Golden Extract: 3:1 or 1:1
mix to prepare a mixture of extracts) mixing ratio 4 One One

<Example 2>

Improvement of rheumatoid arthritis and analysis of therapeutic effects through cell experiments

In order to confirm the improvement and therapeutic effect of rheumatoid arthritis with respect to a total of four types of compositions prepared using herbal medicines of gold and yellow lilies in <Example 1>, rheumatoid arthritis patients (patients satisfying the classification criteria of the American Rheumatology Society in 1987) For the synovial cell line (Fibroblast like synovium, hereinafter FLS) isolated from It was confirmed through the same experiments.

<2-1> Analysis of synovial cell proliferation inhibitory activity

A synovial cell line (Fibroblast like synovium, hereinafter FLS) isolated from a rheumatoid arthritis patient was subcultured 6 times and used for the experiment. was used, and the cell culture was cultured in an incubator maintained at a temperature of 5% CO ₂ and 37 °C.

The change in proliferation of synovial cells according to the composition treatment of the compounding ratios 1 to 4 was analyzed using IncuCyte® S3 System (Essen bioscience). Specifically, the composition of the compounding ratio 1 to 4 was diluted by 1/2 from the concentration of 500 ug/ml to set the interval for each concentration, and then fluorescence-dyed synovial cell line (5 x 10 ³ pieces/200 μl) was added and cultured together. At the same time as the start of the culture, the cells were observed while photographing the plate every 3 hours, and the experiment was terminated after 72 hours of culture. The composition of the above formulation ratios 1 to 4 was dissolved in 100% DMEM solution, and as a negative control, a solution treatment group in which 2% DMEM was added to the cell culture medium was used, and as a positive control, methotrexate used as a treatment for rheumatoid arthritis ( The group treated with methotrexate: MTX) was used.

The proliferation inhibition analysis of synovial cells was analyzed by measuring cell confluence and fluorescence intensity for fluorescently stained synovial cell lines.

The degree of proliferation (%) of synovial cells was calculated by calculating the relative cell density of the test substance (composition)-treated group when the cell density normally proliferated at 72 hours in culture in the negative control group was 100%. .

In addition, the degree of inhibition of proliferation of synovial cells (%) is the relative inhibition (%) of the test substance-treated group when the cell density after 72 hours of the negative control group is 100% and the inhibition is 0%. It was expressed as a calculated value, and the IC ₅₀ value was measured as the treatment concentration value of the test substance at which the cell density was 50% when the cell density in the negative control was 100%.

As a result, as shown in Figure 1, the mixed extract of the combination ratio 1 showed that the IC ₅₀ value exceeded 0.5 mg/ml, and the mixed extract of the combination ratio 2 had the IC ₅₀ value of 0.5 mg/ml. , the mixture of the extracts of the blending ratio 3 was 0.38 mg/ml, while the mixture of the extracts of the blending ratio 4 was the lowest IC ₅₀ value of 0.25 mg/ml among the four blending ratios, among each composition according to the four blending ratios, It was found that the mixture treatment group of the extract corresponding to the formulation ratio 4 had the best synovial cell proliferation inhibitory activity.

Therefore, through these results, the present inventors found that the therapeutic activity of rheumatoid arthritis may differ depending on the compounding ratio and extraction conditions even if the same herbal material is used. It was found that it can effectively inhibit the abnormal proliferation and activity of synovial cells, which is the cause of

<2-2> Synovial cell migration inhibitory activity analysis

In order to confirm the activity of inhibiting migration of synovial cells with respect to each composition obtained under the four extraction conditions of Example 1, fluorescently stained synovial cells were subjected to a wound healing assay dedicated plate (Incucyte cell migration kit, essen). bioscience) until it became a confluent layer. Thereafter, scratches were formed using a Wound maker (Incucyte cell migration kit, essen nbioscicence), and the composition of the present invention was treated by concentration, and then the cell density was analyzed while photographing the culture plate every 3 hours. The analysis was carried out for 72 hours and was repeated a total of 2 times.

Cell migration analysis was analyzed by measuring the number of cells migrated into the scratched area, and when the cell density in which normal proliferation in the negative control group progressed was 100%, the relative cell density of the test substance-treated group was calculated, and the cells IC ₅₀ values for migration inhibition were analyzed.

As a result, as shown in FIG. 2, the migration of synovial cells into the scratched area also showed that the 1:1 mixture treatment group of golden extract and chrysanthemum extract having the lowest IC ₅₀ value among the four composition ratios. Could know. Specifically, the IC ₅₀ value of the formulation ratio 1 was about 0.38 mg/ml, the IC ₅₀ value of the formulation ratio 2 was about 0.43 mg/ml, and the IC ₅₀ value of the formulation ratio 3 was about 0.18 mg/ml. On the other hand, the IC ₅₀ value of the compounding ratio 4 was found to be about 0.1 mg/ml, which is the lowest value among them.

Through these results, it was found that a 1:1 mixture of golden extract and hwangryeon extract can most effectively inhibit the migration of synovial cells of synovial cells.

<2-3> Expression inhibition activity analysis of inflammatory mediators

Among the compositions prepared under the four extraction conditions through the experiments of Examples <2-1> and <2-2>, 1 of the golden extract and the H. The expression inhibitory activity of the inflammatory mediators was analyzed for the 1:1 mixture. Synovial cells were seeded at 1x10 ⁵ cells/well in a 96-well plate, and cultured overnight. After that, the starvation state was maintained for 24 hours, and then the mixture of the formulation ratio 4 of the present invention was pretreated at a concentration of 0.5 mg/mL for 1 hour, and then LPS (300 mg/mL) and PMA (Propidium Monoazide; 50) ng/mL) and after 4 hours of incubation, cells were collected and cell lysates were obtained.

Then, after extracting RNA from the cell lysate, cDNA was synthesized and the expression level of four target activators, IL-1β, IL-6, CCL-2 and CXCL-2, was measured using an ABI 7500 (Applied Biosystems) instrument. to measure the transcript level.

In addition, the level of IL-6 protein, an inflammatory cytokine, was analyzed using an ELISA kit. During the synovial cell treatment, LPS (300 mg/mL) and PMA (50 ng/mL) were additionally treated, and then after 48 hours of incubation. A cell culture medium was obtained, and the content of IL-6 protein contained in the culture medium was measured using a human IL-6 ELISA kit (cat. KAC1261) and an iMarkTM plate reader (Bio-Rad). In the above experiment, the negative control group was treated with 2% DMSO.

As a result, as shown in FIG. 3, the levels of transcripts of IL-1β, IL-6, CCL-2 and CXCL-2, which are inflammatory mediators significantly increased by LPS treatment, were Upon treatment with a mixture of extracts, it was found to be significantly inhibited. In addition, the ELISA analysis also showed that the production of IL-6 was significantly inhibited.

<Example 3>

Analysis of the improvement and therapeutic effect of rheumatoid arthritis in mice with rheumatoid arthritis by administering a 1:1 mixture of golden extract and ragweed extract

Furthermore, the present inventors found that, through the cell experiment of <Example 2>, for a mixture of 4 (a 1:1 mixture of golden extract and yellow liana extract), which was confirmed to have the best rheumatoid arthritis improvement and treatment effect, collagen-induced The following experiments were performed using rheumatoid arthritis mice.

DBA/1J mice used in the collagen-induced rheumatoid arthritis mouse model are 6-week-old male mice, and are maintained in a specific pathogen free (SPF) laboratory with a temperature of 21-23°C and a relative humidity of 40-45%. bred in a state. The number of experimental animals per cage was 6 or less, and cage exchange and feed were provided twice a week. To induce rheumatoid arthritis, dissolve bovine type II collagen to a concentration of 2 mg/mL in 10 mM acetic acid, mix it with CFA (Complete Freund's adjuvant) 2 mg/mL and 1:1 (v/v) to emulsify, and then emulsify it with DBA Primary immunization was induced by intradermal injection at 100 μl/mouse into the tail of /1J mice. After 21 days, IFA (incomplete Freund's adjuvant) and bovine type II collagen were mixed in 1:1 (v/v) to emulsify, and then, 100 μl/mouse was injected intradermally into the tail of the mouse to induce secondary immunization, thereby preventing rheumatoid arthritis. An induced mouse animal model was prepared and used in the following experiments. In addition, all animal experiments performed in the present invention were performed under the approval of the Animal Experimental Ethics Committee (IACUC).

<3-1> Analysis of clinical indicators and frequency of rheumatoid arthritis

After dissolving the mixture according to the formulation ratio 4 of the present invention to the rheumatoid arthritis-induced mice in a PBS solution, it was orally administered once a day from 22±2 days of breeding. At this time, the negative control group was orally administered with PBS solution once a day from 22±2 days, and the positive control group was intraperitoneally injected with MTX twice a week. Treatments for each group of mice used in the experiment are as described in Table 2 below.

Classification of experimental groups processing material route of administration drug dosage drug volume (ul) negative control (vehicle) PBS oral- N/A 200 positive control (control) MTX (methotrexate) intraperitoneal injection 1 mg/kg 100 test group Mixture according to the blending ratio 4 of the present invention oral- 166.5 mg/kg/time 200

The rheumatoid arthritis clinical index (CAI) for each experimental group was given a score of 0-4 according to the following criteria by regularly observing the occurrence and degree of inflammation in the feet of mice until the end of the experiment. , and the sum was used as the clinical activity index.

score standard 0 No arthritis One One inflamed digit 2 Two inflamed digits 3 More than two digits and foot pad inflammation 4 All digits and foot pad inflammation

Criteria for determining clinical indicators

In addition, the disease incidence was judged to have occurred when the clinical index was 2 or higher, and was defined as 100% incidence when arthritis occurred in all four paws of the mouse.

As a result of the analysis, as shown in Figure 4, the negative control mice showed that the symptoms of rheumatoid arthritis worsened and the clinical index values increased over time, whereas the positive control group administered with MTX had the clinical index values compared to the negative control group. appeared to decrease. In addition, the group administered with the mixture of compounding ratio 4 of the present invention also showed a decrease in clinical indicator values compared to the negative control group, and it was found that the clinical indicator values were further reduced compared to the group administered with MTX.

In addition, as a result of the frequency analysis, the incidence of rheumatoid arthritis in the negative control group increased rapidly until the 33rd day, while the positive control group administered with MTX showed a decrease in the incidence compared to the negative control group. The mixture administration group of the compounding ratio 4 of the present invention showed a more remarkably reduced disease incidence compared to the MTX administration group.

In addition, the control group showed severe swelling of the feet in the photographic results of observing the condition of the mouse feet (lesion site) of each experimental group, whereas the MTX-administered group and the mixture treatment group of the present invention with a combination ratio of 4 decreased swelling compared to the control group. Thus, it was confirmed that the symptoms of rheumatoid arthritis were improved (FIG. 5).

These results suggest the possibility that a 1:1 mixture of the golden extract of the present invention and the H. rhubarb extract of the present invention can effectively treat rheumatoid arthritis even through an animal experimental model.

<3-2> Analysis of the therapeutic effect of rheumatoid arthritis through histomorphological analysis

After collecting the tissues of the hind paws from each group of mice tested in <3-1>, each tissue was fixed with paraffin, prepared in sections, and stained with hematoxylin and eosin Morphological evaluation was performed. Morphological evaluation was evaluated according to the criteria in Table 3 below, and 4 sites for each tissue were photographed at 100x magnification, the average value of the assessors' cancer blindness evaluation data was used as the scoring value, and the average of the scoring values of each site was used as the scoring value of each tissue. was calculated as a score.

Histomorphological evaluation items and evaluation criteria

As a result of the analysis, for the symptoms of synovial hyperplasia, pannus formation, cartilage destruction, and bone erosion, in the negative control group, hyperproliferation of synovial cells (synovial fluid proliferation) and With the increase of pannus tissue, cartilage destruction and bone erosion were clearly observed. In the MTX-administered group, the positive control group, the proliferation of synovial cells was weakly observed, but compared to the negative control group, the cartilage and bone destruction were clearly reduced. It also appeared to have decreased. The mixture treatment group of the extract according to the formulation ratio 4 of the present invention also showed a statistically significant decrease in all four evaluation items, and this treatment effect was found to be almost similar to that of the MTX treatment group (see FIG. 6 ).

In addition, the present inventors performed toluene blue staining on the tissues of the mouse group used in the experiment, and analyzed matrix staining, surface regularity and cartilage thickness, and each item Star evaluation criteria were analyzed according to the criteria in Table 5 below.

Histomorphological evaluation items and evaluation criteria

As a result of the analysis, as shown in FIG. 7 , the group administered with a mixture of gold and yellow lotus extract according to the formulation ratio 4 of the present invention, including the positive control group, showed a statistically significant decrease in symptoms compared to the negative control group. Therefore, these results show that a 1:1 mixture of the golden extract of the present invention and the extract of yellow lily is usable as a therapeutic agent for rheumatoid arthritis.

<3-3> Analysis of inflammatory cell infiltration inhibition activity in rheumatoid arthritis tissue through immunostaining

For the paw tissues of mice of each experimental group used in Example <3-2>, the effect of a mixture of gold and yellow lily extract according to the formulation ratio 4 of the present invention on the expression of CD3, F4/80 and ICAM-1 was analyzed. It was analyzed whether inflammatory cell infiltration and the severity of rheumatoid arthritis could be improved. For CD3, F4/80 and ICAM-1 analysis, immunohistochemical staining was performed using these antibodies, and the antibodies used in the experiment were Rabbit anti-CD3 (DAKO, A0452), Rat anti-F4/ 80 (Bio-Rad, MCA497GA) and goat anti-ICAM-1 (R&D, AF796) were used, and as secondary antibodies, Goat anti-rabbit IgG-biotinylated (Vector Lab, BA-1000), Rabbit anti-rat IgG- Biotinylate (Vector Lab, BA-4001) and Rabbit anti-goat IgG-biotinylated (DAKO, E0466) were used. Three or more slides for each tissue were prepared and used for the experiment, observed under a microscope, and the number of cells stained with CD3 or F4/80 antibody and the area stained with ICAM-1 were quantitatively evaluated.

As a result, as shown in FIG. 8, it was found that inflammatory cell infiltration was inhibited by treatment with a 1: 1 mixture of golden extract and yellow liana extract according to the formulation ratio 4 of the present invention, that is, T cells (CD3-positive cells) and It was found that macrophages (F4/80 positive cells) and cells expressing ICAM-1 were significantly reduced in the group treated with a mixture of MTX and a combination ratio of 4 of the present invention compared to the negative control group.

These results mean that a 1:1 mixture of golden extract and yellow liana extract according to the formulation ratio 4 of the present invention can prevent or treat rheumatoid arthritis through inhibition of infiltration of inflammatory cells.

<3-4> Expression inhibition activity analysis of inflammatory mediators through RT-PCR

The joint tissue of each mouse group tested in Example <3-3> was pulverized using ceramic beads to obtain a tissue lysate, and RNA was extracted from the lysate. cDNA was prepared using a cDNA synthesis kit (TAKARA, #6110A) using 3μg of RNA from each experimental group, and the cDNA was reacted with TaqMan Gene Expression Master Mix (ThermoFisher, #4369016), and ABI 7500 (Applied Biosystems) was Transcript levels of target genes IL-6, IL-1β and TNF-α were analyzed in real time using the In addition, the probes used for RT-PCR for measuring the transcript levels of IL-6, IL-1β and TNF-α in the above experiment were Mm00446190_m1 (IL-6), Mm00434228_m1 (IL-1β) and Mm00443258_m1 (TNF-α), respectively. ) was used.

As a result, as shown in FIG. 9, the transcript levels of IL-6, IL-1β and TNF-α were significantly reduced in the MTX-treated group and the mixture-treated group according to the compounding ratio 4 of the present invention compared to the negative control group. appeared, and the inhibitory activity of IL-6, IL-1β and TNF-α was found to be superior to the MTX treatment group in the mixture treatment group of the compounding ratio 4 of the present invention.

Therefore, as described above, through cell and animal experiments, the present inventors found that a mixture of golden extract and yellow liana extract in a weight ratio of 1:1 inhibits proliferation and migration of synovial cell lines, and suppresses the expression of inflammatory mediators By confirming that it is possible to suppress the infiltration of inflammatory cells and improve and treat the symptoms of arthritis, the mixture of the golden extract of the present invention and the H. was found to be available.

So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (7)

Preparing each of the golden extract and hwangryeon extract; and
A method for preparing a mixture of golden extract and yellow lily extract having an improving or therapeutic effect on rheumatoid arthritis, comprising the step of mixing the golden extract and the yellow lotus extract in a weight ratio of 1:1. According to claim 1,
The method for preparing a mixture of gold extract and yellow lily extract having an improving or therapeutic effect on rheumatoid arthritis, characterized in that the gold extract or yellow lily extract is a water extract obtained using purified water. 3. The method of claim 2,
The golden extract or yellow lotus extract is filtered by adding purified water 10 to 12 times the weight of dried gold or yellow lotus and heating at a temperature of 100 to 120 ° C. for 2 to 4 hours. After filtering, at 50 to 60 ° C. A method for preparing a mixture of golden extract and yellow lotus extract having an effect of improving or treating rheumatoid arthritis, characterized in that it is concentrated under reduced pressure to obtain a golden extract or a yellow lily extract. A pharmaceutical composition for the prevention or treatment of rheumatoid arthritis, comprising as an active ingredient a mixture of the golden extract and hwangryeon extract prepared by the method of claim 1 . 5. The method of claim 4,
The mixture of the golden extract and the extract
inhibition of proliferation and migration of synovial cell lines;
inhibition of the expression or production of the inflammatory cytokines IL-6, IL-1β, TNF-α, CCL2 and CXCL2;
inhibition of infiltration of immune cells into joint sites;
inhibition of pannus formation; and
inhibition of cartilage destruction and bone erosion; A pharmaceutical composition for preventing or treating rheumatoid arthritis, characterized in that it has activity. A health functional food for the prevention or improvement of rheumatoid arthritis, comprising as an active ingredient a mixture of golden extract and hwangryeon extract prepared by the method of claim 1 . 7. The method of claim 6,
The mixture of the golden extract and the extract
inhibition of proliferation and migration of synovial cell lines;
inhibition of the expression or production of the inflammatory cytokines IL-6, IL-1β, TNF-α, CCL2 and CXCL2;
inhibition of infiltration of immune cells into joint sites;
inhibition of pannus formation; and
inhibition of cartilage destruction and bone erosion; Health functional food for the prevention or improvement of rheumatoid arthritis, characterized in that it has activity.

Images