KR102763563B1 - Composition for preventing or treating rheumatoid arthritis comprising Pinellia ternata, Wolfiporia extensa, Magnolia Officinalis, Perilla frutescens var. acuta and Zingiber officinale as active ingredients - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating rheumatoid arthritis, comprising a composite extract of Pinellia chinensis, Poria cocos, Magnolia chinensis, Perilla frutescens and ginger as an effective ingredient. The composite extract comprising Pinellia chinensis, Poria cocos, Magnolia chinensis, Perilla frutescens and ginger according to the present invention has all of the activities of inhibiting the proliferation and migration of synovial cell lines, and inhibiting the expression or production of inflammatory cytokines IL-6, IL-1β, TNF-α, CCL2 and CXCL2. As a composite extract derived from natural medicinal herbs, it has no side effects and can therefore be usefully used in the manufacture of pharmaceuticals and health functional foods for effectively preventing, improving and treating rheumatoid arthritis.

Description

{Composition for preventing or treating rheumatoid arthritis comprising Pinellia ternata, Wolfiporia extensa, Magnolia Officinalis, Perilla frutescens var. acuta and Zingiber officinale as active ingredients}

The present invention relates to a composition for preventing or treating rheumatoid arthritis, comprising a composite extract of Pinellia ternata , Wolfiporia extensa , Magnolia Officinalis , Perilla frutescens var. acuta , and ginger ( Zingiber officinale ) as an effective ingredient.

Rheumatoid arthritis (RA) is an autoimmune disease that causes various clinical manifestations through the interaction of genetic, environmental, and immunological factors. In particular, it can be said to be a complex genetic disease in which multiple genes, not just one, are involved.

Rheumatoid arthritis is a chronic systemic inflammatory disease that affects joints, and is pathologically characterized by synovial hypertrophy, inflammatory cell infiltration, and joint destruction. It is known that T cells are activated by an immune response in the early stages, which initiates an inflammatory response. However, as the disease progresses to the final stage, the influence of T cells decreases, and joint destruction continues due to the activation of cartilage-degrading enzymes and bone-degrading factors caused by the abnormal proliferation and activation of synovial cells (synoviocytes). The cells present in the synovium are very diverse, including lymphocytes, macrophages, monocytes, fibroblasts, neutrophils, NK cells, and mast cells. The occurrence of rheumatoid arthritis is complexly caused by the interaction of these various cells and inflammatory cytokines (TNF-α, IL-1, IL-6, IL-17, etc.), and this is known to be related to various symptoms of rheumatoid arthritis.

Symptoms of rheumatoid arthritis include inflammation and infiltration of immune cells, and formation of new blood vessels in the synovium of the peripheral joints such as the fingers, toes, and wrists, causing synovitis, and destruction of articular cartilage and bone damage, resulting in functional impairment and deformation of the joints. In addition to joint symptoms, it may also involve organs (lungs, kidneys, heart), osteoporosis, and vasculitis, and typically symptoms such as pain, stiffness, and swelling in multiple joints appear gradually over several weeks. In about two-thirds of patients, fatigue, loss of appetite, general weakness, and vague musculoskeletal symptoms precede the development of synovitis.

Meanwhile, anti-inflammatory steroids (e.g., prednisolone), non-steroidal anti-inflammatory drugs (e.g., indomethacin, aspirin), immunosuppressants [e.g., cyclosporin A, tacrolimus (FK506), methotrexate, cyclophosphamide, azathioprine], and disease-modifying antirheumatic drugs (e.g., gold salt preparations) are used as treatments for rheumatoid arthritis.

However, anti-inflammatory drugs can control inflammation and alleviate some symptoms, but it is difficult to stop the progression itself, and non-steroidal anti-inflammatory drugs cannot prevent cartilage loss in the joint area or the progression of the disease, and long-term use can cause digestive side effects such as gastric and duodenal ulcers. Steroid hormones are also drugs that control inflammation, but they have side effects such as a round face, weight gain, diabetes, and high blood pressure.

Therefore, most of the drug treatments for rheumatoid arthritis developed to date are used only for the purpose of reducing pain, and there are currently no drugs or surgical methods that provide fundamental treatment effects. Therefore, it is necessary to develop a new treatment for rheumatoid arthritis that does not cause side effects and has excellent preventive and therapeutic effects.

Accordingly, the inventors of the present invention conducted research to develop a new treatment agent that can effectively prevent, improve or treat rheumatoid arthritis without side effects using natural herbal medicines, and as a result, confirmed that a composite extract using the herbal medicines of Pinellia sinensis, Poria cocos, Magnolia nutmeg, Perilla frutescens and ginger can effectively improve and treat rheumatoid arthritis, thereby completing the present invention.

KR 10-1271054

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating obesity, which contains a composite extract of Banha, Poria, Magnolia chinensis, Perilla leaf, and ginger as an effective ingredient.

Another object of the present invention is to provide a health functional food for preventing or improving rheumatoid arthritis, which contains a composite extract of Banha, Poria, Magnolia chinensis, Perilla leaf, and ginger as an effective ingredient.

In order to achieve the above object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising a composite extract of pinellia, Poria, Magnolia chinensis, perilla leaf and ginger as an effective ingredient.

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

In one embodiment of the present invention, the composite extract may be obtained by adding purified water in an amount 10 to 12 times the weight of the composite to a composite of dried pinellia, Poria cocos, Magnolia nutmeg, Perilla frutescens, and ginger, heating the resulting extract at a temperature of 100 to 120°C for 2 to 4 hours, filtering the resulting extract, and then concentrating the extract under reduced pressure at 50 to 60°C.

In one embodiment of the present invention, the composite extract may have an activity of inhibiting proliferation and migration of synovial cell lines; and inhibiting expression or production of inflammatory cytokines IL-6, IL-1β, TNF-α, CCL2, and CXCL2.

In addition, the present invention provides a health functional food composition for preventing or improving rheumatoid arthritis, comprising a composite extract of Banha, Poria, Magnolia chinensis, Perilla leaf, and ginger as an effective ingredient.

The complex extract comprising Pinellia sinensis, Poria cocos, Magnolia chinensis, Perilla frutescens and ginger according to the present invention has all of the activities of inhibiting the proliferation and migration of synovial cells, and the expression or production of inflammatory cytokines IL-6, IL-1β, TNF-α, CCL2 and CXCL2. As a complex extract derived from natural medicinal herbs, it has no side effects in the body, and therefore can be usefully used in the manufacture of medicines and health functional foods for effectively preventing, improving and treating rheumatoid arthritis.

Figure 1 shows the results of the proliferation inhibition effect when the complex extract of the present invention and methotrexate (MTX), a rheumatoid arthritis treatment drug, are respectively treated on a synovial cell line (FLS) isolated from a rheumatoid arthritis patient.
Figure 2 shows the results of analyzing cell proliferation inhibition activity through analysis of cell density and fluorescence intensity at each concentration of methotrexate (MTX) and the complex extract of the present invention.
Figure 3 shows the results of analyzing the inhibition of synovial cell migration by measuring cell density at each concentration of methotrexate (MTX) and the complex extract of the present invention.
Figure 4 shows the results of analyzing the RNA levels of the genes IL-6, IL-1β, and CCL2, which are inflammatory mediators, when treated with the complex extract of the present invention.
Figure 5 shows the ELISA results of IL-6, an inflammatory mediator, when treated with the complex extract of the present invention.

Hereinafter, the present invention will be described in detail with reference to the attached drawings as implementation examples of the present invention. However, the following implementation examples are presented as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and this may vary depending on the intention of the user or operator, or the customs of the field to which the present invention belongs. Therefore, the definition of these terms should be determined based on the contents throughout this specification. Throughout the specification, when a part is said to “include” a certain component, this does not mean that other components are excluded, but rather that other components can be included, unless specifically stated otherwise.

The present invention is characterized by providing a pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising a composite extract of Banha, Poria, Magnolia chinensis, Perilla leaf, and ginger as an effective ingredient.

Banhahoobak-tang is a representative prescription in Oriental medicine that removes bile, a pathological product of stagnant water and body fluid in the digestive tract, which flows back and stagnates in the throat and esophagus, and improves metabolism. It is a compound prescription composed of five natural ingredients: Pinellia sinensis, Poria cocos, Magnolia dentata, Jasoyeop, and Ginger. Currently, Banhahoobak-tang itself is relatively well known for its effect in improving various digestive disorders, including dysphagia, as well as mental disorders.

However, there has been no study reported yet on the effectiveness of Banhahubaktang in treating rheumatoid arthritis.

Accordingly, the inventors of the present invention confirmed through in vitro experiments using cell lines that the composite extract of pinellia, Poria cocos, Magnolia chinensis, perilla leaf, and ginger, which are the constituent ingredients of Banhahoobak-tang, have preventive, improving, or therapeutic effects on rheumatoid arthritis.

The above is a perennial herb of the Araceae family in the order Araceae, growing in fields and growing to a height of about 30 cm. The root is toxic, but it is known to be highly effective for expectoration, diuresis, and headaches due to its effective ingredients such as ephedrine and choline. It also has an antitussive effect that stops coughing, and a gastrointestinal function regulating and phlegm-relieving effect. It is also famous for its effect of stopping vomiting, so much so that it is known as a sacred medicine for the elderly.

The above Poria cocos is a mushroom in the family Poria cocos of the order Amanitas in the order Basidiomycetes, and it parasitizes on the roots of trees such as pine trees underground. The size of the sclerotia is 10–30 cm, and it is round, elongated, or lumpy. The surface is reddish brown, light brown, or blackish brown and rough, and sometimes the root skin is torn. The flesh is white and gradually turns light red. The white one is called white Poria cocos (白茯笭), and the red one is called red Poria cocos (赤茯笭). Also, Poria cocos with pine roots running through it are called Boksin (茯神). Both are herbal medicines that are effective in strengthening, diuresis, and sedation, and are used for kidney disease, cystitis, and urethritis.

The above-mentioned Machilus thunbergii has been used as a stomachic for a long time in Korea, Japan, and China, and the Machilus thunbergii leaves have also been used as food in Korea and Japan. It is known that the Machilus thunbergii tree contains alpha, beta-eudesmol, magnolol, honokiol, and obovatol. It is an evergreen tree of the Lauraceae family in the Ranunculaceae order of dicotyledons, and in oriental medicine, the bark is called Houbakpi (厚朴皮) and is used for asthma and gastrointestinal diseases.

The above-mentioned leaf refers to the leaf of the annual plant of the family Lamiaceae, Chajogi. The stem is square and straight, and the height is 20-80cm. The leaf is ovate, similar to perilla, but purple, has a strong fragrance, and has serrations on the edges. In oriental medicine, the leaf is called Jasoyeop, and the root and the whole plant are used for anemia, sweating, fever reduction, pain relief, bronchitis, seasickness, asthma, phlegm, gastroenteritis, digestion promotion, dizziness and body pain, nasal congestion and runny nose, and as an antidote for fish poisoning.

The above ginger is also called saengang or saeyang, the rhizome grows sideways, is fleshy, lumpy, yellow, has a spicy taste and fragrant smell, and a false stem made of a leaf sheath from each node of the rhizome stands straight up and reaches a height of 30 to 50 cm, the leaves are arranged in two rows on the upper part, the leaves are alternate, linear and lanceolate, both ends are narrow and the bottom part is a long leaf sheath. It does not bloom in Korea, but in tropical regions, a flower stem 20 to 25 cm long wrapped in a leaf sheath comes out in August, and a flower spike grows at the end of it and flowers bloom. The flowers come out between the bracts, are 4 to 7.6 cm long, the receptacle is short and tubular, the end of the corolla is divided into three, and the tips of the divided pieces are pointed. In Korea, it is recorded as a medicinal plant in the Goryeo Dynasty literature, Hyangyak Gugeupbang, and the rhizome is dried and ground and used as a spice in bread, cookies, curry sauce, pickles, etc. It is also pickled in syrup after being peeled and cut, and is also used to make ginger tea and ginger wine. In oriental medicine, the dried rhizome is used as a medicinal herb called ganjang (乾薑), which is effective for indigestion, vomiting, and diarrhea, promotes blood circulation, and has anti-inflammatory and analgesic effects.

The present invention provides a composition for preventing or treating rheumatoid arthritis using a composite extract obtained by mixing and extracting the above-mentioned Banha, Poria, Magnolia chinensis, Perilla leaf, and ginger.

On the other hand, there are countless herbal medicines with pharmacological activity, but when using complex herbal medicines, the desired pharmacological activity can only be confirmed through experiments. This is because even extracts from a single plant or a single medicinal crop contain numerous components, and there are many components that have not yet been identified. When different herbal medicines are mixed, the useful components from different plants in the complex may have unexpected chemical reactions, which may inhibit the pharmacological activity, and harmful side effects may occur in the body.

In this respect, the inventors of the present invention screened the pharmacological activities of various combinations of herbal medicines and confirmed that a composite extract of Pinellia chinensis, Poria cocos, Magnolia chinensis, Perilla frutescens, and ginger had the best therapeutic activity against rheumatoid arthritis without inducing cytotoxicity compared to composite extracts composed of other herbal medicines. Therefore, the herbal medicines of Pinellia chinensis, Poria cocos, Magnolia chinensis, Perilla frutescens, and ginger were selected as a new rheumatoid arthritis treatment agent.

In addition, the present invention confirmed that a composite extract using Banha, Poria, Magnolia chinensis, Perilla leaf, and ginger can prevent or treat rheumatoid arthritis through the proliferation inhibition and migration inhibition activities of synovial cells.

Rheumatoid arthritis is characterized by inflammation and proliferation of synovial cells. Synovial cells have the characteristic of proliferating abnormally like cancer cells in patients with rheumatoid arthritis, and abnormally proliferated synovial cells destroy surrounding cartilage and bone, further worsening rheumatoid arthritis. In addition, inflammatory cytokines and chemokines are related to tumor necrosis factor-α (TNF-α) and interleukin-1 beta (IL-1β). In response to these inflammatory cytokines, synovial cells rapidly proliferate and form tissues called pannus, which destroy cartilage and joint bones.

Therefore, the composite extract of Banha, Poria, Magnolia chinensis, Perilla leaf, and ginger of the present invention has the activity of inhibiting the proliferation and migration of synovial cells, and can effectively treat rheumatoid arthritis by inhibiting the expression and production of inflammatory mediators.

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

In addition, it was shown that the therapeutic effect of the complex extract of the present invention is almost at a similar level to that of methotrexate (MTX), which is used as a treatment for rheumatoid arthritis.

Therefore, the inventors of the present invention were able to determine through various experiments that the composite extract of Banha, Poria, Magnolia chinensis, Perilla leaf, and ginger provided in the present invention can be used as a new treatment for rheumatoid arthritis.

The process for producing the above complex extract can be produced according to a conventional method for obtaining an extract from a natural product known in the art, that is, extraction can be performed using a conventional solvent under conditions of conventional temperature and pressure. For example, extraction can be performed using at least one solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, or mixed solvents thereof, and preferably purified water can be used.

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

The above-mentioned manufactured extract can then be filtered, concentrated, or dried to remove the solvent, and filtration, concentration, and drying can all be performed. For example, filtration can be performed using filter paper or a vacuum filter, concentration can be performed using a vacuum concentrator, and drying can be performed using a freeze-drying method, but is not limited thereto.

Preferably, purified water is added in an amount of 10 to 12 times the weight of the complex to a dried compound of Banha, Poria, Magnolia chinensis, Perilla, and Ginger, and the mixture is heated at a temperature of 100 to 120°C for 2 to 4 hours. The resulting extract is filtered and concentrated under reduced pressure at 50 to 60°C, which is used as the complex extract of the present invention.

Meanwhile, in the present invention, the pharmaceutical composition can be manufactured using, in addition to the effective ingredient of the present invention, a pharmaceutically suitable and physiologically acceptable excipient, and the excipient can include an excipient, a disintegrant, a sweetener, a binder, a coating agent, a swelling agent, a lubricant, a glidant, or a flavoring agent.

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

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

The effective ingredient of the present invention may be included in an amount of 0.1 to 30 wt% 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 with another therapy, the dosage of the co-administered compound will vary depending on the type of co-drug used, the specific drug used, the condition to be treated, etc.

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

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

The term "therapeutically effective amount" as used herein means an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue, animal or human, as considered by a researcher, veterinarian, physician or other clinician, including an amount that induces alleviation of symptoms of a disease or disorder being treated. It is obvious to those skilled in the art that the therapeutically effective dosage and frequency of administration of 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 can be adjusted according to various factors including the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of formulation, and the age, weight, general health, sex and diet of the patient, the time of administration, the route of administration and the excretion rate of the composition, the treatment period, and drugs used simultaneously. In the treatment method of the present invention, in the case of adults, the active ingredient of the present invention can be administered once or several times a day at a dosage of 0.001 mg/kg to 1000 mg/kg, but the range is not particularly limited.

Furthermore, the composite extract according to the present invention is composed of natural herbal medicines prescribed in oriental medicine and therefore does not cause side effects or toxicity in the body, so it can be used safely even when taken for a long period of time. Therefore, the composite extract according to the present invention can also be used as a food composition for preventing or improving rheumatoid arthritis.

Therefore, the present invention can provide a health functional food for preventing or improving rheumatoid arthritis, which contains a composite extract of Banha, Poria, Magnolia chinensis, Perilla leaf, and ginger as an effective ingredient.

The food composition of the present invention can be easily utilized as a food that is effective in preventing and improving rheumatoid arthritis, such as a main ingredient, auxiliary ingredient, food additive, functional food, or beverage of a food.

The term “food” herein refers to a natural product or processed product containing one or more nutrients, preferably one that has gone through a certain level of processing to become directly edible, and in its usual sense includes 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, etc. In addition, the foods in the present invention include, but are not limited to, special nutritional foods (e.g., formulated milk, infant and toddler food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g., ramen, noodles, etc.), bread, health supplements, seasoned foods (e.g., soy sauce, soybean paste, red pepper paste, mixed sauce, etc.), sauces, confectionery (e.g., snacks), candies, chocolates, gum, ice cream, processed dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi types, pickled vegetables, etc.), beverages (e.g., fruit drinks, vegetable drinks, soy milk, fermented beverages, etc.), natural seasonings (e.g., ramen soup, etc.). The above food, beverage or food additive can be manufactured by a conventional manufacturing method.

In addition, the above “functional food” refers to a food group or food composition that has been processed to sufficiently express the body’s regulatory functions regarding biodefense rhythm regulation, disease prevention, and recovery, etc., by using physical, biochemical, or bioengineering techniques to provide added value to the food so that the food’s function can be performed and expressed for a specific purpose, and specifically, it may be a health functional food. The above functional food may include food additives that are acceptable in terms of food science, and may further include appropriate carriers, excipients, and diluents that are commonly used in the manufacture of functional foods.

Furthermore, in addition to those described above, a food containing the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., and the above components may be used independently or in combination.

In a food containing the food composition of the present invention, the amount of the effective ingredient according to the present invention may be comprised in an amount of 0.001 wt% to 90 wt% of the total food weight, preferably 0.1 wt% to 40 wt%, and in the case of a beverage, may be comprised in an amount of 0.001 g to 2 g, preferably 0.01 g to 0.1 g, based on 100 ml. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since the effective ingredient does not have any problems in terms of safety, it may be used in an amount greater than the above range, and therefore 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 only intended to illustrate the content of the present invention, and the scope of the invention is not limited by the examples.

<Example 1>

Preparation of a complex extract of Banha, Poria, Magnolia chinensis, Perilla, and Ginger

After mixing 2.00 g of commercially available herbal medicines, including Pinellia ternata, 1.67 g of Poria cocos, 1.00 g of Magnolia Officinalis, 0.67 g of Perilla leaf, and 1.33 g of ginger, purified water was added in an amount 10 times greater than the weight of the composite of five herbal medicines, and extraction was performed at 100°C for 3 hours to obtain a composite extract, which was then filtered to obtain a filtrate, concentrated under reduced pressure at 60°C or lower, and then vacuum- or freeze-dried to prepare a composite dried extract (composite extract) of Pinellia ternata , Poria cocos ( Wolfiporia extensa ), Magnolia Officinalis , Perilla leaf ( Perilla frutescens var. acuta ), and ginger ( Zingiber officinale ) according to the present invention.

<Example 2>

Analysis of the effect of improving and treating rheumatoid arthritis by a combination of extracts of Banha, Poria, Magnolia chinensis, Perilla, and Ginger through cell experiments

In order to confirm the effect of the composite extract obtained from the combination of the medicinal herbs of Pinellia sinensis, Poria cocos, Magnolia chinensis, Perilla frutescens, and Ginger in <Example 1> above and to treat and improve rheumatoid arthritis, the inventors of the present invention conducted the following experiments using fibroblast-like synovium (hereinafter referred to as FLS) isolated from rheumatoid arthritis patients (patients satisfying the 1987 American College of Rheumatology classification criteria). The effects of the composite extract of the present invention on the proliferation and migration of synovial cells and on the expression of inflammatory mediators were confirmed.

2-1. Analysis of the inhibitory activity of synovial cell proliferation on the combined extracts of Banha, Poria, Magnolia chinensis, Perilla leaf, and ginger

Fibroblast-like synovium (FLS) isolated from patients with rheumatoid arthritis were passaged 4 to 7 times and used in the experiment. The culture medium used was DMEM medium containing 10% fetal bovine serum and 2% penicillin/streptomycin. The cells were cultured in an incubator maintained at 0.5% _CO2 and 37℃.

The analysis of the change in the proliferation of synovial cells according to the treatment with the complex extract of the present invention was analyzed using the IncuCyte® S3 System (Essen bioscience). Specifically, the complex extract of the present invention (500 μg/ml) was added to the synovial cell line (5 x 103 cells/200 μl) and mixed, and then the complex of the complex extract of the present invention and the synovial cell line was dispensed into a 96-well plate at 200 μl per well. Thereafter, the plate was photographed every 3 hours from the start of the culture to observe the cell confluence, and the cell confluence was analyzed after 3 days of culture. Here, the complex extract of the present invention was dissolved in a 100% DMEM solution and used. As a negative control group, a group treated with a solution in which 2% DMEM was added to the cell culture medium was used, and as a positive control group, a group treated with methotrexate (MTX), which is a treatment drug for rheumatoid arthritis, was used.

The analysis of inhibition of synovial cell proliferation by treatment with the complex extract of the present invention was performed using a fluorescently stained synovial cell line, and cell proliferation was analyzed through cell confluence and fluorescence intensity.

The degree of proliferation (%) of synovial cells was expressed as the relative cell density of the test substance treatment group calculated when the cell density that normally proliferated after 72 hours of culture in the negative control group was set as 100%.

In addition, the degree of inhibition of synovial cell proliferation (%) was expressed as the relative inhibition (%) of the test substance treatment group calculated when the cell density of the negative control group after 72 hours was set as 100% cell proliferation and inhibition as 0%.

*As a result, as shown in Fig. 1, the cell density analysis result for the synovial cell line showed that the negative control group had proliferated to almost 100% in the cell plate after 72 hours, showing a high cell density, and the positive control group treated with MTX showed a slightly decreased cell density compared to the negative control group, showing a value of about 90%, whereas the group treated with the complex extract of the present invention showed a significant decrease in cell density, unlike the negative and positive control groups, and rather, cell proliferation was shown to be inhibited. As a result of recalculating this as a cell proliferation inhibition rate, the complex extract treated group of the present invention was shown to have an inhibitory activity on synovial cells of about 69.2%, and the MTX treated group was shown to have a value of about 10%.

In addition, the inventors of the present invention set each concentration section for the complex extract of the present invention and MTX, and cultured the synovial cell line together, photographed the culture plate every 3 hours, and analyzed the cell density and fluorescence intensity for up to 72 hours of culture, and analyzed the IC ₅₀ value for cell proliferation inhibition. The IC ₅₀ value for cell proliferation inhibition was analyzed using an analysis program (Softmax pro v5.4, Molecular Devices). The IC ₅₀ value refers to the treatment concentration value of the test substance at which the cell density becomes 50% when the cell density in the negative control group is 100%.

As a result of the IC ₅₀ value analysis for cell proliferation inhibition, as shown in Fig. 2, the MTX treatment group did not have a significant effect on cell proliferation even when treated at a concentration of 3 mg/ml, whereas the complex extract of the present invention was confirmed to inhibit cell density by 50% at a concentration of 0.56 ± 0.12 mg/ml.

Therefore, through these results, the inventors of the present invention were able to determine that the composite extract of Banha, Poria, Magnolia nutmeg, Perilla leaf, and ginger of the present invention can effectively suppress the abnormal proliferation and activity of synovial cells that cause rheumatoid arthritis, and that its effect is also far superior to that of MTX, a rheumatoid arthritis treatment agent.

2-2. Analysis of the inhibitory activity of synovial cell migration for the combined extracts of Banha, Poria, Magnolia chinensis, Perilla leaf, and Ginger

To verify whether the complex extract of the present invention can inhibit the migration of synovial cells, fluorescently stained synovial cells were cultured until they became a confluent layer in a wound healing assay plate (Incucyte cell migration kit, essen bioscience). Thereafter, a scratch was formed using a wound maker (Incucyte cell migration kit, essen bioscicence), and after treating with the complex extract of the present invention at various concentrations, the culture plate was photographed every 3 hours to analyze the cell density. The analysis was performed for 72 hours, and was repeated a total of 4 times.

The analysis of the above cell migration was analyzed by measuring the number of cells that migrated into the scratched area, and when the cell density in which normal proliferation occurred in the negative control group was set as 100%, the relative cell density of the test substance treatment group was calculated, and the IC ₅₀ value for inhibition of cell migration was analyzed.

As a result, as shown in Fig. 3, the group treated with MTX did not have a significant effect on cell proliferation when treated at a concentration of about 3 mg/ml, but showed an excellent cell migration inhibition effect (IC ₅₀ = 0.04±0.02 mg/ml), whereas the complex extract of the present invention showed a concentration-dependent migration inhibition effect (IC ₅₀ = 0.22±0.19 mg/ml) although the inhibition effect was lower than that of MTX.

2-3. Analysis of the inhibitory activity of the expression of inflammatory mediators by the combined extracts of Banha, Poria, Magnolia chinensis, Perilla leaf, and Ginger

The inventors of the present invention analyzed whether the complex extract of the present invention had an inhibitory activity on the expression of inflammatory mediators. To this end, synovial cells were seeded at ^1x105 cells/well in a 96-well plate and cultured overnight. After that, a starvation state was maintained for 24 hours, and then the complex extract of the present invention (0.5 mg/mL) was pretreated for 1 hour, followed by additional treatment with LPS (300 mg/mL) and PMA (Propidium Monoazide; 50 ng/mL). After 4 hours of culture, the cells were collected and cell lysates were obtained.

Then, RNA was extracted from the cell lysate, cDNA was synthesized, and the transcript levels of the four target inflammatory mediators, IL-1β, IL-6, CCL-2, and CXCL-2, were measured using an ABI 7500 (Applied Biosystems) device.

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

As a result, as shown in Fig. 4, the transcript levels of IL-1β, IL-6, CCL-2, and CXCL-2, which are inflammatory mediators significantly increased by LPS treatment, were hardly expressed by the treatment of the complex extract of the present invention, IL-1β was inhibited by more than 50% in the case of IL-6, and CCL2 was inhibited by more than 85% in the case of CCL2. In addition, ELISA analysis also showed that the production of IL-6 was inhibited by more than 90%.

As a result of analyzing the inhibitory activity against rheumatoid arthritis according to the treatment of a complex extract of Pinellia sinensis, Poria cocos, Magnolia nutmeg, Perilla frutescens, and ginger in vitro using a synovial cell line, it was found that the complex extract of the present invention not only exhibits excellent activity in inhibiting the proliferation of synovial cell lines, but can also effectively inhibit both the transcript and protein levels of inflammatory mediators, and thus can be used to prevent, improve, and treat rheumatoid arthritis.

Claims (5)

A pharmaceutical composition for the prevention or treatment of rheumatoid arthritis, comprising a compound extract of Pinellia ternata , Wolfiporia extensa , Magnolia Officinalis , Perilla frutescens var. acuta , and Zingiber officinale as an active ingredient, having an IC ₅₀ value of 0.5 to 0.6 mg/ml for the proliferation of synovial cells. In the first paragraph,
A pharmaceutical composition for preventing or treating rheumatoid arthritis, characterized in that the above complex water extract has an IC ₅₀ value of 0.1 to 0.4 mg/ml for the migration of synovial cells. In the first paragraph,
A pharmaceutical composition for preventing or treating rheumatoid arthritis, characterized in that the above complex extract is obtained by adding purified water in an amount 10 to 12 times the weight of the complex to a complex of dried Banha, Poria, Magnolia nut, Perilla leaf, and ginger, heating the extract at a temperature of 100 to 120°C for 2 to 4 hours, filtering the extract, and then concentrating it under reduced pressure at 50 to 60°C. In the first paragraph,
The above complex water extract is,
Inhibition of proliferation and migration of synovial cell lines;
A pharmaceutical composition for preventing or treating rheumatoid arthritis, characterized by having an activity of inhibiting the expression or production of inflammatory cytokines IL-6, IL-1β, TNF-α, CCL2 and CXCL2. A health functional food composition for preventing or improving rheumatoid arthritis, comprising as an active ingredient a complex extract of Pinellia ternata , Wolfiporia extensa , Magnolia Officinalis , Perilla frutescens var. acuta , and ginger ( Zingiber officinale ), having an IC ₅₀ value of 0.5 to 0.6 mg/ml at a concentration for the proliferation of synovial cells.